Polypeptide and hyaluronic acid coatings

ABSTRACT

The present invention concerns a polyelectrolyte coating comprising at least one polycationic layer consisting of at least one polycation consisting of n repetitive units having the formula (1) and at least one polyanionic layer consisting of hyaluronic acid. The polyelectrolyte coating has a biocidal activity and the invention thus further refers to the use of said polyelectrolyte coating for producing a device, in particular a bacteriostatic medical device, more particularly an implantable device, comprising said polyelectrolyte coating, and a method for preparing said device and a kit.

The present invention concerns a polyelectrolyte coating comprising atleast one polycationic layer consisting of at least one polycationconsisting of n repetitive units having the formula (1) and at least onepolyanionic layer consisting of hyaluronic acid. The polyelectrolytecoating has a biocidal activity and the invention thus further refers tothe use of said polyelectrolyte coating for producing a device, inparticular a bacteriostatic medical device, more particularly animplantable device, comprising said polyelectrolyte coating, and amethod for preparing said device and a kit.

Nosocomial infections (also called health care associated infections),the fourth leading cause of disease in industrialized countries, are amajor health issue. Year by year, implantation of prostheses and medicaldevices is increasing. In the meantime the prevalence of nosocomialinfections related to implants, which are reported in the literature, isconstantly on the rise. It is known that half of all nosocomialinfections worldwide involve a medical device. Accordingly, the mostsignificant hospital-acquired infections, based on frequency andpotential severity, are those related to procedures e.g. surgical siteinfections and medical devices, including urinary tract infection incatheterized patients, pneumonia in patients intubated on a ventilatorand bacteremia related to intravascular catheter use.

Some key factors for the increase of medical-device related infectionsare i) ageing of the population, ii) multiple drug resistance bacteria,iii) poor development of new designed antibiotic molecules designed. Inthe case of medical devices like implants, the surgical site is anattractive target for pathogens and leads to early complications. Toprevent such infections associated with implants, a local treatment forthe first 6 hours post-implantation is of particular interest.

Innovative, bioactive, smart coatings and materials for reducingnosocomial infections are urgently needed to slow this trend.

The alternate deposition of polycations and polyanions on a substrateleads usually to the formation of a coating, called polyelectrolytecoating. Those polyelectrolyte coatings consist of a multilayeredstructure and their thickness increases with the number of depositionsteps. The potential applications of these kind of coatings arewidespread ranging from energy storage devices to anti-fogging coatingsand bioactive substrates. In this latter area, antimicrobial coatingsare receiving extensive attention due to their importance in the fightagainst nosocomial infections.

One strategy for antimicrobial coatings consists in the design ofanti-adhesive coatings to inhibit attachment and growth of pathogens onthe device. These anti-adhesive coatings thus prevent biofilm formation.However, as the growth of the pathogen is not inhibited by this method,the risk of colonization of another surrounding site is high, inparticular in the case of fragile and immunodeficient people.

Another strategy consists in the design of bactericidal coatings. Thesecoatings usually release anti-microbial agents such as antibiotics thatwere incorporated in the coating structure either during its buildup orby diffusion in the coating after buildup. The release of the activecompound can be triggered for example by enzymatic degradation of thecoating or by a pH change. It can also take place naturally due tohydrolysis of one of the film constituent.

These are interesting approaches; however the release profile in situ ofbactericidal coatings should be perfectly controlled to avoid negativeeffects of overdosed delivered drugs. Moreover, in most of the cases,the release is passive which means antimicrobial agents are delivered inthe presence or absence of bacteria. To circumvent these drawbacks,contact-killing strategies could be more advantageous and consist indamaging the bacteria only when they come in contact with the surface ofthe material.

The inventors of the present invention have recently developed apolyelectrolyte coating using poly-L-arginine (PAR) as polycation andhyaluronic acid (HA) as polyanion.

Said coating was used as a powerful surface coating with antimicrobialproperties and with immunomodulatory properties (Özçelik, H. et al.,2015, Adv. Health. Mat, 4: 20126-2036). However, in this strategy, anantimicrobial peptide was further added to efficiently killconcomitantly bacteria, yeast and fungi. Moreover, the poly-L-arginineused was not monodisperse, but the commercial batch of poly-L-arginineused was composed of polymeric chains with different chain lengths and amolecular weight of more than 70 000 (which correspond to polypeptidechains having more than 400 arginine residues).

Contrary to this, in context of the present invention, the inventorsselected well-defined poly-L-arginine, poly-L-lysine or poly-L-ornithinechains with from 10 to 200 residues, to buildup layer-by-layer coatingswith HA as polyanion.

The inventors surprisingly demonstrated that those coatings showed astrong inhibition of bacterial growth of, for example, S. aureus and M.luteus. These results are unexpected and surprising, in particular,because some of these polymers showed biocidal activity in solutionwhereas said biocidal activity was lost when they were used as coatingin the absence of the polyanion HA.

The biocidal activity against M. luteus (see FIG. 5) is furthermoresurprising and unexpected, because M. luteus is hyaluronidase deficient.The inventors thus demonstrated that the biocidal activity of thepolyelectrolyte coating of the invention is independent of thedegradation of the HA layer, which is contrary to prior art coatingsthat require the degradation of the HA layer.

Furthermore, contrary to other coatings, the mechanism responsible forthe biocidal activity of the coating of the present invention seems tobe based on the surface contact of the bacteria with the coating.

The inventors further demonstrated that the polycation molecules diffusewithin the coating and the biocidal activity seems to depend on freediffusion of the polycation molecules, because cross-linking of thecoating reduces its biocidal function (see FIGS. 10 and 11).

SUMMARY OF THE INVENTION

The present invention therefore relates to a polyelectrolyte coating,comprising:

(a) at least one polycationic layer consisting of at least onepolycation consisting of n repetitive units having the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   each R group, identical or different, is chosen from —NH₂,        —CH₂—NH₂ and —NH—C(NH)—NH₂, and        (b) at least one polyanionic layer consisting of hyaluronic        acid.

The present invention relates, in particular, to a polyelectrolytecoating, comprising:

(a) at least one polycationic layer consisting of n repetitive unitshaving the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   R is chosen from —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂, and        (b) at least one polyanionic layer consisting of hyaluronic        acid.

The present invention also relates to a device comprising thepolyelectrolyte coating of the invention.

The present invention further relates to the use of the polyelectrolytecoating of the invention for producing a device of the invention, inparticular a medical device, more particularly an implantable device.

The invention further concerns a method for preparing a devicecomprising the polyelectrolyte coating of the invention, the methodcomprising:

(a) providing a device;

(b1) depositing on the surface of said device

(i) at least one polycationic layer consisting of at least onepolycation consisting of n repetitive units having the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   each R group, identical or different, is chosen from —NH₂,        —CH₂—NH₂ and —NH—C(NH)—NH₂, and then        ii) at least one polyanionic layer consisting of hyaluronic        acid, or        (b2) depositing on the surface of said device ii) and then i) as        defined above, and optionally repeating step b1) and/or b2).

The invention further concerns, in particular, a method for preparing adevice comprising the polyelectrolyte coating of the invention, themethod comprising:

(a) providing a device;

(b1) depositing on the surface of said device

(i) at least one polycationic layer consisting of n repetitive unitshaving the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   R is chosen from —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂, and then        ii) at least one polyanionic layer consisting of hyaluronic        acid, or        (b2) depositing on the surface of said device ii) and then i) as        defined above, and optionally repeating step b1) and/or b2).

In a further aspect, the invention refers to a kit comprising

a) at least one polycationic material consisting of n repetitive unitshaving the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   each R group, identical or different, is chosen from —NH₂,        —CH₂—NH₂ and —NH—C(NH)—NH₂, and        b) at least one polyanionic material consisting of hyaluronic        acid.

In a particular aspect, the invention refers to a kit comprising

a) at least one polycationic material consisting of n repetitive unitshaving the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   R is chosen from —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂, and        b) at least one polyanionic material consisting of hyaluronic        acid.

In a further aspect, the invention refers to a method of preventing abacterial infection in an individual undergoing an implantation of animplantable device comprising the steps of providing an implantabledevice as defined herein below, and implanting said implantable devicein the individual wherein said implantable device prevents a bacterialinfection.

DESCRIPTION OF THE INVENTION

Polyelectrolyte Coating

The inventors of the present inventions have demonstrated that apolyelectrolyte coating with polyarginine, polyornithine or polylysineas polycation and hyaluronic acid (HA) as polyanion is a powerfulsurface coating with biocidal properties. The inventors demonstratedthat, a polyelectrolyte coating with poly-L-arginine (PAR),poly-L-ornithine (PLO) or poly-L-lysine (PLL) with, in particular, 30arginine, ornithine or lysine residues, respectively, and hyaluronicacid (HA) as polyanion has strong biocidal activities.

The inventors further demonstrated that, a polyelectrolyte coating withpolyornithine with 100 ornithine residues and hyaluronic acid (HA) aspolyanion has strong biocidal activity.

The wording “polyelectrolyte”, as known by the skilled in the art,refers to polymers whose repeating units bear an electrolyte group.Polycations and polyanions are both polyelectrolytes. Accordingly, thepolyanionic and polycationic layers in context of the invention may bereferred to as polyelectrolyte layers.

The polycation of formula (1) consists of n repetitive units, saidrepetitive units being identical or different. According to theinvention, the repetitive unit of the polycation has the formula—NH—CH(CH₂—CH₂—CH₂—R)—C(═O). For a given repetitive unit, R is asdefined above and may thus be different for each unit.

According to one preferred embodiment, the polycation of formula (1)consists of n repetitive units wherein all the R groups are identical.

According to a further embodiment, the polycation of formula (1)consists of n repetitive units wherein the R groups may be different.

Among the n units, the polycation may comprise i units of formula—NH—CH(CH₂—CH₂—CH₂—NH₂)—C(═O), j units of formula—NH—CH(CH₂—CH₂—CH₂—CH₂—NH₂)—C(═O), and k units of formula—NH—CH(CH₂—CH₂—CH₂—NH—C(NH)—NH₂)—C(═O), wherein each i, j, and k iscomprised between 0 and n, and wherein i+j+k=n, with a randomdistribution of the units or with a distribution as blocks. The word “atleast” in “at least one polycation” herein refers to at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 polycations consisting of n repetitive units havingthe formula (1), preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 polycations,preferably one polycation consisting of n repetitive units having theformula (1).

“a polycation consisting of n repetitive units having the formula (1)”or only “n repetitive units having the formula (1)” as herein defined isa positively charged polymer and can also be referred to as“polycationic material”. Accordingly, in one embodiment, thepolycationic material of n repetitive units having the formula (1) asdefined in context of the invention constitutes the at least onepolycationic layer of the polyelectrolyte coating of the invention.

In one embodiment, the “n” of the n repetitive units having the formula(1) is an integer comprised between 11 and 100, when R is chosen from—NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂.

In a further embodiment, n is an integer comprised between 11 and 99,for example, n is an integer comprised between 11 and 95, 15 and 95, 15and 90, 15 and 85, 15 and 80, 15 and 75, 20 and 95, 20 and 90, 20 and85, 20 and 80, 20 and 75, 25 and 95, 25 and 90, 25 and 85, 25 and 80, 25and 75, 28 and 74, 28 and 72, 30 and 70, such as 30, 50 and 70, when Ris chosen from —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂, preferably, when R ischosen from —CH₂—NH₂ and —NH—C(NH)—NH₂, more preferably, when R is—NH—C(NH)—NH₂.

In a further embodiment, n is an integer comprised between 11 and 49,for example, n is an integer comprised between between 11 and 45, 15 and45, 20 and 40, 21 and 39, 22 and 38, 23 and 37, 24 and 36, 25 and 35, 26and 34, 27 and 33, 28 and 32, 29 and 31, when R is chosen from —NH₂,—CH₂—NH₂ and —NH—C(NH)—NH₂, preferably, when R is chosen from —CH₂—NH₂and —NH—C(NH)—NH₂, more preferably, when R is —NH—C(NH)—NH₂.

In one particular embodiment, n is an integer selected from the groupconsisting of 11, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 47, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,preferably, n is 30, 50 or 70, when R is chosen from —NH₂, —CH₂—NH₂ and—NH—C(NH)—NH₂, preferably, when R is chosen from —CH₂—NH₂ and—NH—C(NH)—NH₂, more preferably, when R is —NH—C(NH)—NH₂.

In one particular embodiment, n is an integer selected from the groupconsisting of 11, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 45 and 49, preferably, n is 30, whenR is chosen from —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂, preferably, when R ischosen from —CH₂—NH₂ and —NH—C(NH)—NH₂, more preferably, when R is—NH—C(NH)—NH₂.

In one particular embodiment, n is an integer comprised between 11 and150, for example between 11 and 140, 11 and 130, 11 and 120, 11 and 120,15 and 110, 15 and 100, 20 and 100, 22 and 95, 24 and 90, 26 and 80, 26and 75, 26 and 70, 26 and 65, 26 and 55, 26 and 50, 26 and 45, 26 and40, 26 and 35, 26 and 34, 27 and 33, 28 and 32, 29 and 31, when R is—NH₂. In one embodiment, n is an integer selected from the groupconsisting of 11, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 70, 80, 90, 100,110, 120, 130, 140, preferably, n is 30 or 100, when R is —NH₂.

In one embodiment, n is an integer as defined above with the provisothat n is not smaller than 11 and n is not bigger than 49, when R ischosen from —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂, preferably, when R ischosen from —CH₂—NH₂ and —NH—C(NH)—NH₂, more preferably, when R is—NH—C(NH)—NH₂, preferably n is not smaller than 15, 20, or 25 and n isnot bigger than 45, 40, or 35, when R is chosen from —NH₂, —CH₂—NH₂ and—NH—C(NH)—NH₂, preferably, when R is chosen from —CH₂—NH₂ and—NH—C(NH)—NH₂, more preferably, when R is —NH—C(NH)—NH₂.

In one particular embodiment, n is an integer as defined above with theproviso that n is not smaller than 20 or 25 and n is not bigger than 95or 100, when R is —NH—C(NH)—NH₂.

In one particular embodiment, n is an integer as defined above with theproviso that n is not smaller than 20 or 25 and n is not bigger than 45or 40, when R is —NH—C(NH)—NH₂.

In one further particular embodiment, n is an integer as defined abovewith the proviso that n is not smaller than 20 or 25 and n is not biggerthan 45 or 40, when R is —NH₂.

In one further particular embodiment, n is an integer as defined abovewith the proviso that n is not smaller than 20 or 25 and n is not biggerthan 45 or 40, when R is —CH₂—NH₂.

The “repetitive unit” of formula (1) can also be called “structuralunit” and herein refers to an amino acid or amino acid residue, whereinsaid amino acid is ornithine when R is —NH₂, lysine when R is —CH₂—NH₂or arginine, when R is —H—C(NH)—NH₂. Accordingly, “n repetitive units offormula (1)” may also be referred to as “n amino acid residues offormula (1)”, more precisely as n ornithine residues when R is —NH₂, nlysine residues when R is —CH₂—NH₂ or n arginine residues when R is—H—C(NH)—NH₂.

In one embodiment, the n repetitive units of formula (1) polymerize viathe formation of a peptide bond. Accordingly, n repetitive units havingthe formula (1) or n amino acid residues of formula (1) may be referredto as polymer or polypeptide.

In some embodiments, the i, j, and k units as defined herein abovepolymerize via the formation of a peptide bond. Accordingly, thepolycationic material comprising i units of formula—NH—CH(CH₂—CH₂—CH₂—NH₂)—C(═O), j units of formula—NH—CH(CH₂—CH₂—CH₂—CH₂—NH₂)—C(═O), and k units of formula—NH—CH(CH₂—CH₂—CH₂—NH—C(NH)—NH₂)—C(═O), wherein each i, j, and k iscomprised between 0 and n, and wherein i+j+k=n, with a randomdistribution of the units or with a distribution as block, may bereferred to as copolymer.

A “peptide bond”, also called amide bond, is a covalent chemical bondformed between the carboxyl group (COOH) of one amino acid and the aminogroup (NH₂) of another amino acid, wherein one molecule of water isproduced.

According to the above, in some embodiments, “n repetitive units havingthe formula (1)” may be referred to as “polyornithine having n ornithineresidues” when R is —NH₂, “polylysine having n lysine residues” when Ris —CH₂—NH₂ or “polyarginine having n arginine residues” when R is—H—C(NH)—NH₂.

“Ornithine” is a non proteinogenic amino acid that plays a role in theurea cycle. Polyornithine refers to a polymer of the structural unitornithine. Polyornithine refers to poly-L-, poly-D- orpoly-LD-ornithine. In context of the present invention, polyornithinerefers in particular to poly-L-ornithine (PLO).

“Arginine” and “Lysine” are α-amino acids that are used in thebiosynthesis of proteins. Polyarginine and -lysine refer to a polymer ofthe structural unit arginine or lysine, respectively. Polyarginine or-lysine refer to poly-L-, poly-D- or poly-LD-arginine or -lysine. Incontext of the present invention, polyarginine or polylysine refer, inparticular, to poly-L-arginine (PAR) and poly-L-lysine (PLL),respectively.

“Poly-L-ornithine”, “poly-L-lysine” and “poly-L-arginine” are positivelycharged synthetic polymers (also called polycations) and are produced inthe form of a salt with a counterion. The counter ion may be selectedfrom, but is not limited to, hydrochloride, hydrobromide ortrifluoracetate.

In one example, polyarginine is poly-L-arginine hydrochloride with CAS#26982-20-7.

In one example, polyornithine is poly-L-ornithine hydrobromide with CAS#27378-49-0 or poly-L-ornithine hydrochloride with CAS #26982-21-8.

In one example, polylysine is poly-L-lysine triifluoracetate,poly-L-lysine hydrobromide with CAS #25988-63-0 or poly-L-lysinehydrochloride with CAS #26124-78-7.

Poly-L-ornithine, poly-L-lysine and poly-L-arginine having a definednumber of amino acid residues may be obtained commercially, for example,via Alamanda Polymers, USA.

In one example, poly-L-arginine (PAR) such as PAR10 (10 arginine (R),Mw=2.1 kDa, PDI=1); PAR30 (30 R, Mw=6.4 kDa, PDI, =1.01), PAR50 (50arginine (R), Mw=9.6 kDa, PDI=1.03); PAR70 (70 arginine (R), Mw=13.4kDa, PDI, =1.01), PAR100 (100 R, Mw=20.6 kDa, PDI=1.05), and PAR200 (200R, Mw=40.8 kDa, PDI=1.06) were purchased from Alamanda Polymers, USA.

In another example, poly-L-ornithine (PLO) such as PLO30 (30 R, Mw=5.9kDa, PDI=1.03), PLO100 (100 R, Mw=18.5 kDa, PDI=1.03), and PLO250 (250R, Mw=44.7 kDa, PDI=1.02) were purchased from Alamanda Polymers, USA.

In a further example poly-L-lysine (PLL) such as PLL10 (10 R, Mw=1.6kDa), PLL30 (30 R, Mw=5.4 kDa, PDI=1.02), PLL100 (100 R, Mw=17.3 kDa,PDI=1.07), PLL250 (250 R, Mw=39.5 kDa, PDI=1.08) was purchased fromAlamanda Polymers, USA.

Methods to obtain polypeptides having n repetitive units such aspolyarginine, polylysine, or polyornithine with for example n=30 areknown to the skilled in the art and include ring-opening polymerizationof alpha-amino acid N-carboxyanhydrides (NCAs) followed by purification.Typically, the polypeptides are purified after polymerization byprecipitation in water or, for example, in an organic nonsolvent and,after amino acid side chain deprotection, by dialysis. All water-solublepolymers are finally lyophilized.

Methods to obtain copolymers having n units are also known to theskilled in the art.

In one preferred embodiment, the n repetitive units having the formula(1) are monodisperse, i.e. the polycationic material of which thepolycationic layer consists is monodisperse. It will be understood bythe skilled in the art that if the polycationic material of which thepolycationic layer consists is monodisperse, the polycationic layer isas well monodisperse. Accordingly, in one embodiment, the polycationiclayer in context of the present invention is monodisperse.

“Monodisperse” herein refers to a polymer consisting of the samemolecules having the same mass. Synthetic monodisperse polymer chainscan be made, for example, by processes such as anionic polymerization, amethod using an anionic catalyst to produce chains that are similar inlength, for example, ring-opening polymerization of alpha-amino acidN-carboxyanhydrides (NCAs). It can be concluded on the basis of thepolydispersity index (PDI) if a sample of a polymer is monodisperse.Accordingly, in one embodiment, monodispersity is expressed using thepolydispersity index (PDI).

In some embodiments, when the at least one polycation is more than onepolycation, then the at least one polycation may be polydisperse, i.e.the polycationic material of which the polycationic layer consists is amixture of different polycations and may be polydisperse. It will beunderstood by the skilled in the art that if the polycationic materialof which the polycationic layer consists is polydisperse, thepolycationic layer is as well polydisperse. Accordingly, in oneembodiment, the polycationic layer in context of the present inventionis polydisperse.

“Polydisperse” herein refers to a polymer consisting of differentmolecules having a different mass.

The “polydispersity index (PDI)” or “heterogeneity index”, or simply“dispersity”, is a measure of the distribution of molecular mass in agiven polymer sample. The polydispersity index (PDI) is calculated bydividing the weight average molecular weight (M_(w)) with the numberaverage molecular weight (M_(n)). The PDI has a value equal to orgreater than 1, but as the polymer chains approach uniform chain length,the PDI approaches 1.

Accordingly, in one embodiment, the polydispersity index (PDI) issmaller than 1.5, smaller than 1.4, smaller than 1.3, in particularbetween 1 and 1.2, preferably between 1 and 1.1, for example between 1and 1.05.

In one particular embodiment, the polydispersity index (PDI) of the nrepetitive units having the formula (1), the polycationic material orthe polycationic layer is smaller than 1.5, smaller than 1.4, smallerthan 1.3, in particular between 1 and 1.2, preferably between 1 and 1.1,for example between 1 and 1.05.

As known by the skilled in the art the PDI may be measured bysize-exclusion chromatography (SEC), light scattering measurements suchas dynamic light scattering measurements or mass spectrometry such asmatrix-assisted laser desorption/ionization (MALDI) or electrosprayionization mass spectrometry (ESI-MS).

In one example, the PDI is measured either on the protected polyaminoacids by, typically, gel permeation chromatography (GPC) in, forexample, DMF with 0.1M LiBr at typically 60° C. or on the deprotectedpolypeptides by, for example, GPC in typically aqueous buffer using, inboth cases, a calibration curve that was constructed from narrowpolydispersity PEG standards or universal calibration of TALLS. Theaverage molecular weight is provided by TALLS or by proton NMRspectroscopy using the amino acid repeating unit to incorporatedinitiator peaks integration ratio.

“Hyaluronic acid (HA)” also known as Hyaluronan is a linear (unbranched)polysaccharide or non-sulfated glycosaminoglycan, composed of repeatingdisaccharide units of N-acetyl glucosamine and glucuronate (linked by β1-3 and β 1-4 glycosidic bonds). Hyaluronic acid (HA) thus is anegatively charged polymer (also called polyanion) and is therefore, incontext of the present invention, also referred to as polyanionicmaterial. Said negatively charged polymer therefore exists together witha couter ion in form of a salt. For sodium hyaluronate the counterion issodium. It is distributed widely throughout connective, epithelial andneural tissues as part of the extra-cellular matrix. There are highconcentrations in the vitreous and aqueous humor of the eye, synovialfluid, skin, and the umbilical cord (Wharton jelly). The average 70-kgman has roughly 15 grams of hyaluronan in his body, one-third of whichis turned over (degraded and synthesized) every day. It is anevolutionarily conserved molecule being found in both the group A and CStreptococci and Pasteurella multocida as well as birds, mammals, andother orders of animals. In solutions of moderate to high molecularweight (500,000 to >3 million Da) at low concentrations it impartsconsiderable viscosity to aqueous solutions. Hyaluronic acid can bedegraded from Hyaluronidase. The molecular weight (Mw) of hyaluronanrepresents an average of all the molecules in the population and thusrepresents the molecular Mass Average (Molecular Weight Average). In oneexample, Hyaluronic acid has a molecular weight of 150 kDa and isbrought in form of Sodium Hyaluronate from Lifecore Biomed, USA.

“Hyaluronidase” is a family of enzymes that degrade hyaluronan. Theenzyme is found in most animal species and many micro-organisms. Thereare a number of different types of hyaluronidase with differentspecificity and kinetics.

In one embodiment, the polyelectrolyte coating may further comprise a“pharmaceutical active drug”.

In the context of the present specification the term “pharmaceuticalactive drug” refers to compounds or entities which alter, inhibit,activate or otherwise affect biological events. For example, the drugincludes, but is not limited to, anti-cancer substances,anti-inflammatory agents, immunosuppressants, modulators ofcell-extracellular matrix interaction including cell growth inhibitors,anticoagulants, antrithrombotic agents, enzyme inhibitors, analgetic,antiproliferative agents, antimycotic substances, cytostatic substances,growth factors, hormones, steroids, non-steroidal substances, andanti-histamines. Examples of indication groups are, without beinglimited thereto analgetic, antiproliferativ, antithrombotic,anti-inflammatory, antimycotic, antibiotic, cytostatic,immunosuppressive substances as well as growth factors, hormones,glucocorticoids, steroids, non-steroidal substances, genetically ormetabolically active substances for silencing and transfection,antibodies, peptides, receptors, ligands, and any pharmaceuticalacceptable derivative thereof. Specific examples for above groups arepaclitaxel, estradiol, sirolimus, erythromycin, clarithromycin,doxorubicin, irinotecan, gentamycin, dicloxacillin, quinine, morphin,heparin, naproxen, prednisone, dexamethason.

In one embodiment, the “pharmaceutical active drug” is the polycationicmaterial as defined herein above.

In one embodiment, the polyelectrolyte coating of the invention isbiocompatible.

The term “biocompatible” as used in context of the invention, intends todescribe a coating that does not elicit a substantial detrimentalresponse in vivo.

In one embodiment, the polyelectrolyte coating of the invention hasimmunomodulatory properties.

“Immunomodulatory properties” herein refers to inhibiting thepro-inflammatory pathway.

In other words, in a further embodiment, the polyelectrolyte coating ofthe invention has an inhibitory effect on the production ofpro-inflammatory cytokines.

“Pro-inflammatory cytokines” are produced predominantly by activatedmacrophages and are involved in the up regulation of inflammatoryreactions. In contrast to anti-inflammatory cytokines, which promotehealing and reduce inflammation, pro-inflammatory cytokines act to makea disease worse.

Accordingly, the pro-inflammatory cytokines in context of the inventionare released by immune cells, such as macrophages, in particular, by ahuman primary macrophage subpopulation. Pro-inflammatory cytokines arefor example, but is not limited to, TNF-α, CCL18 and CD206.

Macrophage activation can be divided into two categories. M1macrophages, or classically activated macrophages, which are activeduring initial inflammation, but wherein their long-term presence mayresult in chronic inflammation and M2 macrophages, or alternativelyactivated macrophages, which have a significant role in tissueremodeling and healing. Unbalanced activation of macrophages results inthe development of pronounced and prolonged type 1 (M1). TNF-α is an M1specific cytokine, CCL18 and CD206 are M2 specific cytokines.

As mentioned herein above, the inventors of the present inventiondemonstrated surprisingly that the polyelectrolyte coating of theinvention has biocidal activity.

Accordingly, in one embodiment, the polyelectrolyte coating of theinvention has biocidal activity.

“Biocidal activity” herein refers to destroy, deter, render harmless, orexert a controlling effect on any harmful organism. A biocidal activityherein refers, for example, to an antimicrobial activity.

“Antimicrobial activity” herein refers to antiseptical, antibiotic,antibacterial, antivirals, antifungals, antiprotozoals and/orantiparasite activity, preferably antibacterial activity.

Accordingly, in one embodiment, the polyelectrolyte coating of theinvention has antibacterial activity and/or bacteriostatic activity.

In one embodiment the antibacterial activity and/or bacteriostaticactivity is directed against at least one bacterium.

“Bacteriostatic activity” herein refers to stopping bacteria fromreproducing, while not necessarily killing them, in other wordsbacteriostatic activity herein refers to inhibiting the growth ofbacteria. Accordingly, bacteriostatic activity may be expressed, forexample, in % of growth inhibition of at least one bacterium.

The “growth inhibition of at least one bacterium” in context of thepresent invention, may be more than 70%, for example, more than 75, morethan 80%, typically, more than 82, 84, 86, 88, 90, 91, 92, 93, 94, 95,96, 97, 98%.

Accordingly, in one embodiment, the polyelectrolyte coating of theinvention has more than 70% growth inhibition of at least one bacterium,more particularly, more than 75%, more than 80%, typically, more than82, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98% growth inhibition ofat least one bacterium.

The “at least one bacterium” herein refers to bacteria of at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more species of bacteria.

In one embodiment, the at least one bacterium is a ESKAPE pathogen.

The “ESKAPE pathogens” are the leading cause of nosocomial infectionsthroughout the world and are described in, for example, Biomed Res Int.2016; 2016: 2475067. In one embodiment, the term “ESKAPE pathogens”refers to a bacterium selected from the group constituted ofEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterspecies.

In one embodiment, the at least one bacterium is a gram-positivebacterium or gram-negative bacterium, preferably gram-positivebacterium.

In one embodiment, the gram-negative bacterium is a Pseudomonasaeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia,Escherichia coli, Klebsiella pneumoniae, Enterobacter species orLegionella bacterium, preferably, Escherichia coli or Pseudomonasaeruginosa

In one embodiment, the gram positive bacterium is a Staphylococcus,Micrococcus or Enterococcus bacterium.

Bacteria of the “Staphylococcus” genus are stationary,non-spore-forming, catalase-positive, oxidase-negative, gram-positivecocci grouped together in grape-like clusters. Observed by Pasteur in1879 in furuncle pus, staphylococci owe their name to Ogsten (1881) whoisolated them in acute chronic abscesses. Bacteria of the“Staphylococcus” genus, such as, for example, S. aureus, S. epidermidis,S. capitis, S. caprae, S. haemolyticus, S. lugdunensis, S. schleiferi,S. simulans and S. warneri are the main agents of infections on foreignmaterials for example in prosthetic joint infections.

Accordingly, in one embodiment the Staphylococcus is selected from S.aureus, S. epidermidis, S. capitis, S. caprae, S. haemolyticus, S.lugdunensis, S. schleiferi, S. simulans and S. warneri, preferably S.aureus and S. epidermidis, more preferably S. aureus.

Bacteria of the “Micrococcus” genus are generally thought to be asaprotrophic or commensal organism, though it can be an opportunisticpathogen, particularly in hosts with compromised immune systems, such asHIV patients. Micrococci are normally present in skin microflora, andthe genus is seldom linked to disease. However, in rare cases, death ofimmunocompromised patients has occurred from pulmonary infections causedby Micrococcus. Micrococci may be involved in other infections,including recurrent bacteremia, septic shock, septic arthritis,endocarditis, meningitis, and cavitating pneumonia in particular inimmunosuppressed patients.

In one embodiment the Micrococcus is a M. luteus bacterium.

Bacteria of the “Enterococcus” genus are the cause of important clinicalinfections such as urinary tract infections, bacteremia, bacterialendocarditis, diverticulitis, and meningitis.

In one embodiment the Enterococcus is a vancomycin-resistantEnterococcus, such as E. faecalis or E. faecium.

The bacteriostatic activity or % of growth inhibition may bedemonstrated, for example, in an antibacterial assay as herein describedin the section “methods” herein below. Strains that may be used in suchan antibacterial assay may be, for example, M. luteus or S. aureus.

As mentioned in the introduction herein above, in one typicalapplication, the polyelectrolyte coating of the present inventionparticularly aims at preventing nosocomial infections related toimplants and medical devices. In said context, the risk of an infectionis especially high during the 6 hours post-implantation.

Accordingly, in one embodiment, the polyelectrolyte coating of thepresent invention has a bacteriostatic activity within the first 24 hrspost implantation, for example within the first 12 hrs, first 9 hrs,first 6 hrs post implantation.

As further mentioned in the introduction, antibacterial coatings have avast field of applications. Accordingly, in one embodiment thepolyelectrolyte coating of the invention has an anti-fouling activity.

“Fouling” or “Biofouling” or “biological fouling” herein refers to theaccumulation of microorganisms on a wetted surface.

“Anti-fouling” therefore herein refers to inhibiting the accumulation ofmicroorganisms on a wetted surface.

The polyelectrolyte coating of the present invention is typicallyconstructed using a layer-by-layer (LbL) deposition technique as furtherdescribed in the section “Method for preparing a device” herein below.Based on the layer-by-layer structure the polyelectrolyte coating mightalso be referred to as a “polyelectrolyte multilayer (PEMs)”,“polyelectrolyte film” or “polyelectrolyte matrix”.

Each of the polyelectrolyte layers has its given charge. Thepolycationic layer and the polyanionic layer, both form apolyelectrolyte network or a polyelectrolyte backbone. Thepolyelectrolyte layers attract each other by electrostatic interactions.Other attractive forces are based on hydrophobic, van der Waals, andhydrogen bonding interactions.

In general, during LbL deposition, a device, such as, for example, amedical device or an implantable device is dipped back and forth betweendilute baths of positively and negatively charged polyelectrolytesolutions. During each dip a small amount of polyelectrolyte is adsorbedand the surface charge is reversed, allowing the gradual and controlledbuild-up of electrostatically cross-linked polycation-polyanion layers.It is possible to control the thickness of such coatings down to thesingle-nanometer scale.

Accordingly, the polyelectrolyte coating of the invention typicallyincludes substantially ordered polyelectrolyte layers of alternatinglycharged polyelectrolyte layers.

In one embodiment, a single polyelectrolyte layer, such as onepolycationic or polyanionic layer, has a thickness of 1 nm to 10 nm. Thethickness of the polycationic and/or polyanionic layer depends on thecoating conditions and the polyelectrolyte material used.

In one embodiment, the polyelectrolyte coating has a thickness which istypically substantially thicker than the thickness of a singlepolyelectrolyte layer of the polyelectrolyte coating. In one embodiment,the polyelectrolyte coating may have a thickness of about 10 nm to about100000 nm. The thickness of the polyelectrolyte coating depends on thecoating conditions, the number of, and the polyelectrolyte material usedfor, the polyelectrolyte layers.

The thicknesses of an obtained polyelectrolyte coating may be evaluated,for example, using confocal microscopy. Therefore, for example, 100 μLof PLL-FITC (poly-L-lysine labeled with fluorescein isothyocyanate, agreen fluorescent probe) (typically 0.5 mg·mL⁻¹ in Tris-NaCl buffer) aredeposited on top of a polyelectrolyte coating, for example a(PAR30/HA)₂₄ polyelectrolyte coating. After 5 minutes and diffusion ofPLL-FITC through the whole polyelectrolyte coating, a rinsing step istypically performed with Tris-NaCl buffer. Observations of the coatingsmay be carried out with a confocal microscope, such as Zeiss LSM 710microscope (Heidelberg, Germany) using a 20× objective (Zeiss, PlanApochromat).

As described above, the polyelectrolyte coating may be formed in aself-assembled manner to produce a Layer-by-Layer (LbL) structure.

The term “layer” in “polycationic layer” or “polyanionic layer” hereinrefers to an amount of polycations or polyanions as defined hereinabove, for example, deposited on, for example, the surface of a device,wherein said device is as defined herein below in the section “device”and may be, preferably, a medical device or an implantable device. As itwill be understood by the skilled in the art, in context of theinvention, one polycationic layer may consist of several layers of thesame polycationic material, and one polyanionic layer may consist ofseveral layers of the same polyanionic material.

In some embodiments, “at least one polycationic layer” and/or “at leastone polyanionic layer” in context of the invention refers to at least 1,5, 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 200, 300, 400, 500 polycationic layers and/orpolyanionic layers.

In some embodiments, “at least one polycationic layer” and/or “at leastone polyanionic layer” in context of the invention refers to at least 1,5, 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60polycationic layers and/or polyanionic layers.

In some embodiments, “at least one polycationic layer” and/or “at leastone polyanionic layer” refers to 1 to 500 polycationic and/orpolyanionic layers, for example 1 to 400, 1 to 300, 1 to 200, 1 to 100,1 to 90, 1 to 80, 1 to 70, 1 to 60, 5 to 60, 10 to 60, 20 to 60,preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60 layers.

In some embodiments, “at least one polycationic layer” and/or “at leastone polyanionic layer” refers to 1 to 100 polycationic and/orpolyanionic layers, for example 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to50, 1 to 40, 1 to 35, 1 to 30, such as 5 to 40, 10 to 40, 15 to 40, 20to 40 layers, preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 layers.

In certain embodiments the number of the polycationic layers and thenumber of the polyanionic layers are the same.

In certain embodiments, the polycationic and polyanionic layers arealternating layers, in particular alternating charged polyelectrolytelayers.

Accordingly, in one embodiment, the polyelectrolyte coating comprises 1to 60 polycationic layers and/or 1 to 60 polyanionic layers.

In a particular embodiment, the polyelectrolyte coating comprises 18 to60 polycationic layers and/or 18 to 60 polyanionic layers, morepreferably the polyelectrolyte coating comprises 18 to 50 polycationiclayers and/or 18 to 50 polyanionic layers.

In a particular embodiment, the polyelectrolyte coating comprises 18 to60 polycationic layers and/or 18 to 60 polyanionic layers, morepreferably the polyelectrolyte coating comprises 18 to 40 polycationiclayers and/or 18 to 40 polyanionic layers.

In one example, as further described herein below, it is possible tocover the surface of an object with one polycationic layer, to wash theobject and cover it again with one polycationic layer. These steps canbe repeated several times in order to obtain one polycationic layer of aspecific thickness which thus consists of several polycationic layers.As it will be understood by the skilled in the art this procedure can beused as well to obtain one polyanionic layer of a certain thicknesswhich is thus constituted of several polyanionic layers.

In one example, the polyelectrolyte coating consists of 24 layers ofpolyarginine having 30 arginine residues (PAR30) and 24 layers of HA,accordingly said coating will herein be called (PAR30/HA)₂₄. In the sameexample, the first layer is a polycation layer consisting of PAR30,followed by a first layer of the polyanion HA, followed by a secondpolycation layer consisting of PAR30 and followed by a second polyanionlayer consisting of HA. The layers are alternating until the 24^(th)polycation layer consisting of PAR30 and the 24^(th) polyanion layerconsisting of HA.

In another example, the polyelectrolyte coating consists of 48 layers ofpolyarginine having 30 arginine residues (PAR30) and 24 layers of HA,accordingly said coating will herein be called (PAR30/HA)₄₈. In the sameexample, the first layer is a polycation layer consisting of PAR30,followed by a first layer of the polyanion HA, followed by a secondpolycation layer consisting of PAR30 and followed by a second polyanionlayer consisting of HA. The layers are alternating until the 48^(th)polycation layer consisting of PAR30 and the 48^(th) polyanion layerconsisting of HA.

In a further aspect of the invention, the polyelectrolyte coating mayalso, for example, start with a polyanionic layer on the surface of anobject and finish with a polyanionic layer, in that case the number ofthe polycationic and polyanionic layers are different. Preferably, thepolyelectrolyte coating starts with a polyanionic layer when the surfaceof the object is positively charged.

In another aspect of the invention, the polyelectrolyte coating mayalso, for example, start with a polycationic layer on the surface of anobject and finish with a polycationic layer; in that case the number ofthe polycationic and polyanionic layers is as well different.Accordingly, in certain embodiments the number of the polycationiclayers and the number of the polyanionic layers are different.Preferably, the polyelectrolyte coating starts with a polycationic layerwhen the surface of the object is negatively charged.

In one example, the polyelectrolyte coating consists of 25 layers ofpolyarginine having 30 arginine residues and 24 layers of HA,accordingly said coating will be called (PAR30/HA)₂₄-PAR30.

In certain embodiments, different polyelectrolyte layers consist of thesame polycationic material or of different polycationic materials asherein defined. For example, the polyelectrolyte coating may comprise 12layers consisting of polyarginine having 30 arginine residues and 12layers consisting of polyornithine having 30 ornithine residues and 24layers consisting of HA.

The inventors of the present invention observed an exponential growth ofthe normalized frequency with the number of deposition steps for thebuild-up of the polyelectrolyte coatings with PAR30, PAR100 or PAR200and HA using quartz crystal microbalance (QCM), as further explained inthe examples and FIG. 1. The inventors further demonstrated that theexponential increase of the thickness with the number of depositing stepis related to the diffusion, in and out of the whole coating, of atleast one polyelectrolyte constituting the multilayer. The inventorsfurther demonstrated using bleaching experiments that the polycationicpolymer contained in the coating is mobile and thus diffuses inside thewhole coating, as demonstrated in FIG. 14.

Accordingly, in one embodiment, the polyelectrolyte coating of theinvention raised an “exponential growth”, as called in the literature,of the normalized frequency with the number of deposition steps.

In a further embodiment, the n repetitive units having the formula (1)as define herein above are mobile and/or diffuse within thepolyelectrolyte coating.

It has been shown by the inventors, that the covalent coupling of the atleast two oppositely charged polyelectrolyte layers reduces thebacteriostatic activity of the coating.

Accordingly, in one embodiment the at least one polycationic layer andthe at least one polyanionic layer are not covalently coupled.

Device

Staphylococcal infections on foreign material differ from conventionalinfections by the arrangement of bacteria in the form of biofilm.

“Biofilm” is a complex three-dimensional structure which is connected tothe foreign material and in which the bacteria cells are embedded in apolysaccharide extracellular matrix called slime or glycocalyx. Thisspecific structure may be formed by bacteria of the same species or ofdifferent species. In comparison with their living congeners in free (or‘planctonic’) form, these bacteria are in a state of quiescenceindicated by a low level of metabolic activity. Due to the reducedmetabolic activity bacteria in the form of a biofilm are, for example,more resistant to any antibacterial treatment.

The problematic of biofilms is not limited to the medical field.Biofilms are ubiquitous, occurring in aquatic and industrial watersystems as well as a large number of environments. Biofilms can avidlycolonize the surfaces of a wide variety of household items such astoilet bowls, sinks, toys, cutting boards, and countertops in kitchenand bathrooms. Biofilms may also occur in the food production industry,either in tins or on the machines that are used along the productionlines. At water and sewage treatment facilities, biofilms (biofouling)are also problematic: they cause metal corrosion, increased risk ofcontamination of products, decreased quality of water, and reducedefficacy of heat exchange for example for boards, and countertops inkitchen and bathroom.

Accordingly, an antibacterial coating is an advantage for devices inmany different applications.

As described above, the inventors of the present invention developed apolyelectrolyte coating having a biocidal activity, in particular withinthe first 72 hours when contacted with a solution containing bacteria,in particular within the first 48 hours when contacted with a solutioncontaining bacteria, more preferably the first 24, first 12, first 6hours when contacted with a solution containing bacteria. Thepolyelectrolyte coating of the present invention therefore inhibitsgrowth of a bacterium and thus prevents bacteria from the formation of abiofilm of the surface of a device comprising the polyelectrolytecoating of the invention.

The inventors demonstrated, for example, that (PAR/HA)24 coatings builtwith PAR30, PAR50, PAR100 and PAR30 after 24/48 or 72 h of incubationshow a total inhibition of bacteria which demonstrate their efficiencyover 3 days and three successive contaminations. The inventors furtherdiscovered that the time period of the biocidal activity of the coatingsof the invention increases with the number of layers. Accordingly, thetime period of the biocidal activity of the coating of the presentinvention increases with the number of layers used.

The inventors further demonstrated that neither drying nor sterilizationof the coatings of the invention did modify the antimicrobial activityof the coating. Accordingly no change in the total bactericide activitywas measured even after sterilization.

It will be therefore understood by the skilled in the art that due tothe bacteriostatic activity of the polyelectrolyte coating of theinvention, said polyelectrolyte coating is, in particularly suitable forproducing a device comprising said polyelectrolyte coating.

Accordingly, the present invention further refers to a device comprisinga polyelectrolyte coating of the invention.

A “device” herein refers to an object comprising at least one surface.

In one embodiment, the polyelectrolyte coating covers at least a portionof the surface of said device.

In one embodiment, the surface of the device of the present inventioncomprises, consists of, or at least partly consists of metal such astitanium, plastic such as silicone, ceramic or other materials such aswood.

In a further embodiment, the surface of the device of the presentinvention exists in any kind of architecture depending on the materialused, accordingly, it will be understood by the skilled in the art, thatthe surface may be a flat surface, or un uneven surface, such an unevensurface exists, for example, on surfaces of porous materials such astypically foams or fibers. In some examples, the surface may alsocomprise or consist of micro or nanoparticles.

Since the device comprises the polyelectrolyte coating of the invention,it will be understood by the skilled in the art, that, therefore, thefeatures associated with said polyelectrolyte coating that are furtherdefined above in the section “polyelectrolyte coating” also refer tosaid device.

Accordingly, in one embodiment, the device of the invention has biocidalactivity, wherein the biocidal activity is as defined in the section“polyelectrolyte coating” herein above.

In one aspect of the invention, the device of the invention has morethan 70% growth inhibition of at least one bacterium, more particularly,more than 75%, more than 80%, typically, more than 82, 84, 86, 88, 90,91, 92, 93, 94, 95, 96, 97, 98% growth inhibition of at least onebacterium. % growth inhibition of at least one bacterium is as definedherein above in the section “polyelectrolyte coating”.

In a further embodiment, the device of the present invention has abacteriostatic activity within the first 72 hours post implantation, forexample within the first 48 hours, 24 hours, 12 hours, first 9 hours,and first 6 hours post implantation.

In a preferred embodiment, the device of the present invention has abacteriostatic activity within the first 24 hours post implantation, forexample within the first 12 hours, first 9 hours, and first 6 hours postimplantation.

Furthermore, in one embodiment the device of the invention hasanti-fouling activity. In a further embodiment, the device of theinvention has antibacterial activity and/or bacteriostatic activity.

Accordingly, in one embodiment, the device comprising a polyelectrolytecoating of the invention is a bacteriostatic device and/or a foulingresistant device. Bacteriostatic is as defined herein above in thesection “polyelectrolyte coating”.

The polyelectrolyte coating of the invention and the device of theinvention can be easily sterilized and stored without detrimentaleffects to the coating and its properties.

It will be further understood by the skilled in the art that due to thebacteriostatic activity of the polyelectrolyte coating of theinventions, said polyelectrolyte coating is, in one embodimentparticularly suitable for producing a medical device, preferably aimplantable device.

A “medical device” herein refers to an instrument, apparatus, implement,machine, contrivance, implant, in vitro reagent, or other similar orrelated article, including a component part, or accessory which is forexample intended for use in the diagnosis of disease or otherconditions, or in the cure, mitigation, treatment, or prevention ofdisease, in an individual as defined herein below, or intended to affectthe structure or any function of the body of an individual, and whichdoes not achieve any of its primary intended purposes through chemicalaction within or on the body of man or other animals and which is notdependent upon being metabolized for the achievement of any of itsprimary intended purposes. In one example, a medical device refers to adevice used for wound healing, such as bandages, for example adhesivebandages or dressings. In a further example, a medical device refers tosanitary articles.

A medical device herein may be destinated for use in an individual.

The term “individual”, “patient” or “subject” refers to a human ornon-human mammal, preferably a mouse, cat, dog, monkey, horse, cattle(i.e. cow, sheep, goat, buffalo), including male, female, adults andchildren.

Accordingly, in one embodiment, the medical device in context of theinvention may be a medical instrument.

In one embodiment, a medical instrument may be selected from the groupconsisting of a percussion hammer, pleximeter, thermometer, foreign bodydetector, stethoscope, specula, forceps, otoscope, or any accessoriesthereof, probes, retractors, scalpel, surgical scissors, boneinstruments, sharps spoons, suture needles and wounds clips.

In one embodiment, the medical device is an implantable device.

The “implantable device” of the present invention refers to a piece ofequipment or a mechanism that is placed inside of the body of anindividual to serve a special purpose or perform a special function.

An implantable device, in context of the present invention, may be, forexample, a prosthetic device or, for example, a device implanted todeliver medication, nutrition or oxygen, monitor body functions, orprovide support to organs and tissues. Implantable devices may be placedpermanently or they may be removed once they are no longer needed. Forexample, stents or hip implants are intended to be permanent. Butchemotherapy ports or screws to repair broken bones can be removed whenthey no longer needed.

In one embodiment, the implantable device is selected from the groupcomprising catheters, arteriovenous shunts, breast implants, cardiac andother monitors, cochlear implants, defibrillators, dental implants,maxillofacial implants, middle ear implants, neurostimulators,orthopedic devices, pacemaker and leads, penile implants, prostheticdevices, replacement joints, spinal implants, voice prothesis,artificial hearts, contact lenses, fracture fixation device, infusionpumps, intracranial pressure device, intraocular lenses, intra-uterinedevices, joint prosthesis, prosthetic valves, orthopedic devices, suturematerials, urinary stents, vascular assist device, vascular grafts,vascular shunts and vascular stents, and artificial vessels of permanentor transient types.

In a preferred embodiment, the implantable device is selected from thegroup comprising catheters, defibrillators, prosthetic devices,prosthetic valves, replacement joints, orthopedic devices, pacemakers,vascular grafts, vascular shunts, vascular stents and intra-uterinedevices, preferably catheters, orthopedic devices, pacemakers andprosthetic devices.

A “catheter” herein refers to a tubular medical device for insertioninto canals, vessels, passageways, or body cavities for diagnostic ortherapeutic purposes, fluids and medication, but also in drainage ofbody fluids such as urine or abdominal fluids; angioplasty, angiography,and catheter ablation; administration of gases such as oxygen andvolatile anesthetic agents and hemodialysis.

A “shunt” typically refers to a narrow metal or plastic tube thatdiverts blood from one part to another.

A “stent” typically refers to a short narrow metal or plastic tube oftenin the form of a mesh that is inserted into the lumen of an anatomicalvessel as an artery or bile duct especially to keep a previously blockedpassageway open.

A “prosthetic valve” is for example a prosthetic heart valve.

Method for Preparing a Device of the Invention

The present invention further refers to a method for preparing a deviceof the invention herein referred to as the method of the invention.

The device of the invention may comprise different materials asspecified herein above, accordingly the at least one surface of thedevice to be covered with the polyelectrolyte coating may comprisedifferent materials as specified herein above. It will be understood bythe skilled in the art that said materials differ in their surfacecharge. Positively charged surfaces are, for example, the surfacesconsisting of amine group based polymers. Negatively charged surfacesare for example the surfaces consisting of carboxylic groups basedpolymers. It will be further understood by the skilled in the art, thata positively charged surface will be first covered with a polyanioniclayer and then with a polycationic layer, wherein a negatively chargedsurface will be first covered with a polycationic layer and then with apolyanionic layer. The two consecutive deposition steps may then berepeated as often as needed.

In one example, an device of the invention is prepared by depositing on,for example, its SiO₂ surface 24 bilayers of PAR/HA (PAR30/HA)₂₄ using,for example, an automated dipping robot, such as the an automateddipping robot of Riegler & Kirstein GmbH, Berlin, Germany. Therefore,the surface of the device is typically first washed with, for example,Hellmanex® II solution at 2%, H2O, and ethanol and dried with air flow.Solutions of polyelectrolytes such as PAR and HA are prepared, forexample, by dissolving PAR and HA at typically 0.5 mg·mL⁻¹ in sterilizedbuffer containing typically 150 mM NaCl and, for example, 10 mM oftris(hydroxymethyl)-aminomethan (TRIS, Merck, Germany) at, typically, pH7.4. The surface of the device is dipped alternatively in polycation andpolyanion solutions and extensively rinsed in NaCl-Tris buffer betweeneach step. After preparation, the coating is, typically, dried with airflow and then immerged in NaCl-Tris buffer and stored at 4° C. beforeuse.

The wording “depositing on the surface of said device at least onepolycationic layer” and “depositing on the surface of said device atleast one polyanionic layer” of step b1) i) and ii) or b2) ii) and i)herein refers to contacting the surface of said device with apolycationic solution in case of step b1) i) or b2 i) or to contactingthe surface of said device with a polyanionic solution in case of stepb1) ii) or b2 ii).

The “the surface of said device” herein refers to at least one surface,said at least one surface may be partially covered by thepolyelectrolyte coating of the invention. The at least one surface ispreferably one surface.

A “polycationic solution” or “polyanionic solution” herein refers to asolution comprising polycationic material or polyanionic material asdefined herein above in the section “polyeletrolyte coating”. In contextof the present invention, typically a “polycationic solution” and a“polyanionic solution” might be referred to as polyelectrolyte solution.

“Contacting” as herein used refers typically to spraying, immersing,dipping or pouring.

Accordingly, in one embodiment, for “depositing the polyelectrolytelayers according to step (b1) and/or (b2)”, a polyelectrolyte solutionmay, for instance, be sprayed onto the surface of a device on which thepolyelectrolyte coating is to be formed.

Alternatively, or in combination, the surface of said device can beimmersed or dipped into a polyelectrolyte solution or a polyelectrolytesolution can be poured onto the surface of the substrate.

Accordingly, in one embodiment, the at least one polycationic layer isdeposited on the surface of the device in step b1) i) by contacting thesurface of said device with a polycationic solution comprising nrepetitive units having the formula (1) as defined herein above.

Accordingly, in one embodiment, the at least one polyanionic layer isdeposited on the surface of the device in step b1) ii) by contacting thesurface of said device with a polyanionic solution comprising HA.

It will be understood that the steps b1) and/or b2) may be repeated asoften as necessary by the skilled in the art in order to obtain a devicecomprising a polyelectrolyte coating with the number of polyelectrolytelayers, in particular alternating polycationic and polyanionic layers,as defined herein above in the section “polyelectrolyte coating”. Itwill be clear to the skilled in the art, that the deposition of thepolyelectrolytes may be influenced by the pH.

Accordingly, in one embodiment, said “polycationic solution” and/or“polyanionic solution” in context of the invention may further comprisea buffer.

A “buffer” is an aqueous solution consisting of a mixture of a weak acidand its conjugate base or a weak base and its conjugate acid. Its pHchanges very little when a small amount of strong acid or base is addedto it and thus it is used to prevent changes in the pH of a solution.Buffer solutions are used as a means of keeping pH at a nearly constantvalue in a wide variety of chemical applications. Buffers used in thecontext of the invention might be, for example, PBS (Phosphate bufferedsaline) or TRIS (tris(hydroxymethyl)aminomethane).

In one embodiment, said “polycationic solution” and/or “polyanionicsolution” in context of the invention has pH ranging from 4 to 9,preferably 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 and typically, a pHranging from 5 to 8, 5.5 to 8, 6 to 8, 6.5 to 8, 7 to 8, preferably 7 to8, in particular 7, 7.2, 7.4, 7.6, 7.8, 8, for example 7.4.

It will be further clear to the skilled in the art, that the depositionof the polyelectrolytes may be influenced by the interaction between thecharged polyelectrolytes and the charged surface onto which thepolyelectrolytes are to be deposited, and the interaction between thecharged polyelectrolytes among each other. These interactions can be atleast partially controlled by the ion strength of the solution.

Therefore, in some embodiments the ion strength of the polyelectrolytesolution, used in step (b1) or (b2) of the method of the invention isadjusted in certain embodiments to increase the amount of the depositedpolyelectrolytes.

Accordingly, in one embodiment, said “polycationic solution” and/or“polyanionic solution” in context of the invention may further compriseions.

An “ion” is an atom or molecule in which the total number of electronsis not equal to the total number of protons, giving the atom or moleculea net positive (cation) or negative (anion) electrical charge. An ionconsisting of a single atom is an atomic or monatomic ion; if itconsists of two or more atoms, it is a molecular or polyatomic ion. Ionsare further distinct according to the charge they carry. Therefore anion can be a monovalent ion or bivalent (sometimes called divalent ion)or polyvalent ions. Ions may be without limitation F⁻, Cl⁻, I⁻, NH₄ ⁺,SO₄ ²⁺, Ca²⁺, Mg²⁺, Na⁺.

Typically, ions may added in the form of a buffer, as defined hereinabove, or in the form of a salt, such as for example NaCl.

For example, solutions of polyelectrolytes such as PAR and HA weretypically prepared, for example, by dissolving PAR and HA at typically0.5 mg·mL-1 in sterilized buffer containing typically 150 mM NaCl and,for example, 10 mM of tris(hydroxymethyl)-aminomethan (TRIS, Merck,Germany) at, typically, pH 7.4.

Between the depositions of the polyelectrolyte layers at least onewashing or also called rinsing step can be performed in a solutionwithout polyelectrolytes to remove not-assembled polyelectrolytematerial.

Accordingly, in one embodiment, the method of the invention furthercomprises at least one washing step after step step i) and/or ii) of b1)or b2).

In one embodiment, for washing, a solution without polyelectrolytes maybe used. Said solution may be water or any other solution that seemssuitable to the skilled in the art. In one embodiment, washing isperformed using buffer, for example NaCl-Tris buffer. In one example,said NaCl-Tris buffer comprises 150 mM NaCl and 10 mM Tris at a pH 7.4.

Alternatively, or in addition to that, in one embodiment, a drying stepor steps can be performed.

Accordingly, in one embodiment, the method of the invention furthercomprises at least one drying step after step i) and/or ii) of b1) orb2) and/or the washing step of claim.

“Drying” may be performed by any method known to the skilled in the art,such as air heating, natural air drying, dielectric drying, air flow,preferably air flow. In one example air flow refers to purging withnitrogen gas.

In one embodiment, after a drying step, the same polyelectrolytesolution as used immediately before the drying step can be depositedagain on the surface since the drying can lead to a partial exposure ofbinding sides or charges of the underlying polyelectrolyte layer, saiddrying step may therefore also be called an intermediate drying step. Byapplying such a sequence, the load of a particular polyelectrolyte, canbe further increased.

Accordingly, in one embodiment when the method of the invention furthercomprises at least one drying step the previous step i) or ii) of b1) orb2) and/or the washing step may be repeated.

Typically, a polyelectrolyte solution is brought into contact with thesurface and the surface is allowed to dry for a given time, for examples10 to 60 sec by purging, for example, with nitrogen gas. Subsequently,the same polyelectrolyte solution is again brought into contact with thesurface.

In certain embodiments, at least one washing and/or drying step iscarried out between two consecutive deposition steps.

It has been shown by the inventors, that the covalent coupling of the atleast two oppositely charged polyelectrolyte layers reduces thebacteriostatic activity of the coating.

Accordingly, in one embodiment the method of the invention does notcontain a step to covalently couple the at least one polycationic layerwith the at least one polyanionic layer.

In one embodiment, the method of the invention further comprises a step(c) wherein the polyelectrolyte coating obtained in steps (a) and (b1)or (b2) of the method as defined above may be further soaked with apharmaceutical active drug. The pharmaceutical drug is as defined in thesection “Polyelectrolyte coating” herein above.

In one particular embodiment, the method for preparing a device is amethod for method for preparing an implantable device.

Accordingly, in one embodiment, the invention refers to a method forpreparing an implantable device comprising a polyelectrolyte coating,the method comprising:

(a) providing an implantable device;

(b1) depositing on the surface of said implantable device

(i) at least one polycationic layer consisting of at least onepolycation consisting of n repetitive units having the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   each R group, identical or different, is chosen from —NH₂,        —CH₂—NH₂ and —NH—C(NH)—NH₂, and then        ii) at least one polyanionic layer consisting of hyaluronic        acid, or        (b2) depositing on the surface of said implantable device ii)        and then i) as defined above, and optionally repeating step b1)        and/or b2).

Accordingly, in a particular embodiment, the invention refers to amethod for preparing an implantable device comprising a polyelectrolytecoating, the method comprising:

(a) providing an implantable device;

(b1) depositing on the surface of said implantable device

(i) at least one polycationic layer consisting of n repetitive unitshaving the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   R is chosen from —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂, and then        ii) at least one polyanionic layer consisting of hyaluronic        acid, or        (b2) depositing on the surface of said implantable device ii)        and then i) as defined above, and optionally repeating step b1)        and/or b2).

As described herein above under the section “device”, the surface of thedevice of the present invention comprises, consists of, or at leastpartly consists of metal such as titanium, plastic such as silicone,ceramic or other materials such as wood. These surfaces may be charged,such as positively or negatively charged. As a result, the skilled inthe art will therefore understand that a device comprising a surfacethat is, for example, negatively charged might be covered first with acationic layer.

In some embodiments, the device might undergo a surface treatment priorto the method for preparing an implantable device in order to have acharged surface.

Accordingly, the surface of the device provided in step a) might becharged, such as positively charged or negatively charged, wherein thesurface is as defined herein above in the section “device”.

Alternatively, in some embodiments the method for preparing animplantable device might comprise a step (a2) of charging the surface ofsaid implantable device.

The skilled in the art knows methods to charge the surfaces of a device,such methods include the adsorption of ions, protonation/deprotonation,and the application of an external electric field. The skilled in theart further knows, for example, that surface charge practically alwaysappears on a device surface when it is placed into a fluid. Most fluidscontain ions, positive (cations) and negative (anions). These ionsinteract with the device surface. This interaction might lead to theadsorption of some of them onto the surface. If the number of adsorbedcations exceeds the number of adsorbed anions, the surface would have anet positive electric charge, and if the number of adsorbed anionsexceeds the number of adsorbed cations, the surface would have a netnegative electric charge.

Use

In one embodiment the invention refers to the use of the polyelectrolytecoating of the invention for producing a device of the invention, inparticular a fouling resistant device and/or bacteriostatic device.

In a further embodiment the invention refers to the use of thepolyelectrolyte coating of the invention for producing a medical deviceor an implantable device.

Kit

The invention further provides a kit comprising

a) at least one polycationic material consisting of n repetitive unitshaving the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   each R group identical or different is chosen from —NH₂,        —CH₂—NH₂ and —NH—C(NH)—NH₂, and        b) at least one polyanionic material consisting of hyaluronic        acid.

In particular, the invention further provides a kit comprising

a) at least one polycationic material consisting of n repetitive unitshaving the formula (1),

wherein

-   -   n is an integer comprised between 11 and 100, and    -   R is chosen from —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂, and        b) at least one polyanionic material consisting of hyaluronic        acid.        In one embodiment, the kit further comprises instructions        regarding the use of the polycationic and polyanionic material.        These instructions may e.g. describe a method for preparing an        implantable device as defined herein above.

“n”, “R” and the “repetitive units having the formula (1)” are asdefined herein above in the section “polyelectrolyte coating”.

The wording “at least” in “at least one polycationic material” or in “atleast one polyanionic material” herein refers to at least 1, 2, 3, 4, 5,6, 7, 8 polycationic materials.

In one embodiment, the kit comprises

-   -   a) at least one polycationic material consisting of polyarginine        with 11 to 95, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to 75,        20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 25 to 95, 25        to 90, 25 to 85, 25 to 80, 25 to 75, 28 to 74, 28 to 72, 30 to        70 arginine residues, preferably poly-L-arginine, more        preferably, poly-L-arginine with 30 L-arginine residues, 50        L-arginine residues, or 70 L-arginine residues, and/or    -   b) at least one polycationic material consisting of polylysine        with 11 to 99 arginine residues, preferably with 11 to 95, 15 to        90, 20 to 85, 20 to 80, 25 to 75, 25 to 70, 25 to 65 25 to 60,        25 to 55, 25 to 50, 25 to 40, 25 to 35, preferably        poly-L-lysine, more preferably, poly-L-lysine with 30 L-lysine        residues, and/or    -   c) at least one polycationic material consisting of        polyornithine with 11 to 150 ornithine residues, preferably with        11 to 140, 11 to 130, 11 to 120, 11 to 120, 15 to 110, 15 to        100, 15 to 95, 15 to 90, 15 to 80, 15 to 75, 20 to 70, 10 to 65,        20 to 55, 20 to 50, 20 to 45 ornithine residues, preferably        poly-L-ornithine, more preferably, poly-L-ornithine with 30        L-lysine residues, and    -   d) at least one polyanionic material consisting of hyaluronic        acid.        In one embodiment, the kit comprises    -   a) polycationic material consisting of polyarginine with 15 to        45 arginine residues, preferably poly-L-arginine, more        preferably, poly-L-arginine with 30 L-arginine residues, and/or    -   b) polycationic material consisting of polylysine with 15 to 45        lysine residues, preferably poly-L-lysine, more preferably,        poly-L-lysine with 30 L-lysine residues, and/or    -   c) polycationic material consisting of polyornithine with 15 to        120 ornithine residues, preferably poly-L-ornithine, more        preferably, poly-L-ornithine with 30 L-lysine residues, and        at least one polyanionic material consisting of hyaluronic acid.        Therapeutic Methods and Uses

Infections, such as Staphylococcal infections, on foreign materialassociated with biofilm formation have a number of features whichdistinguish them completely from conventional tissue infections. Theseinfections are most often paucibacillary (having few bacteria) andreadily polymicrobial. The bacteria have a very slow metabolism whichkeeps them in a state close to dormancy and the genes which they expressare different to those activated in planctonic forms. The state ofdormancy of the bacteria and the presence of the biofilm significantlyreduce the inflammatory reaction and the attraction of immune cells atthe infection site. Lastly, for the same reasons, the bacteria arelargely protected from the action of antibiotics. It is thereforeimportant to prevent said biolfilm formation, in particular, in the bodyof an individual, for example after transplantation.

The polyelectrolyte coating or implantable device in context of thepresent invention have a biocidal activity, in particular within thefirst 72 hours post-implantation, in particular within the first 48hours post-implantation, more preferably the first 24, first 12, first 6hours post implantation. The polyelectrolyte coating or implantabledevice in context of the present invention therefore inhibit growth of abacterium and thus prevents them from the formation of a biofilm of thesurface of said coating or implantable device and thus prevents abacterial infection.

Accordingly, in one embodiment, the invention refers to a method ofpreventing a bacterial infection in an individual undergoing animplantation of an implantable device comprising the steps of providingan implantable device as defined herein above, and implanting saidimplantable device in the individual, wherein said implantable deviceprevents a bacterial infection.

In one embodiment, providing an implantable device refers to preparingan implantable device according to the method of the invention.

The individual is as defined herein above in the section “device”.

In one embodiment, the bacterial infection is a nosocomial infection.

A “nosocomial infection” also called Hospital-acquired infection (HAI)herein refers an infection that is contracted from the environment orstaff of a healthcare facility. The infection is spread to thesusceptible patient in the clinical setting by a number of means, inparticular by implantable devices. Gram positive bacteria involved in“nosocomial infection” are for example Staphylococcus aureus.Gram-negative bacteria involved in “nosocomial infection” are forexample Pseudomonas aeruginosa, Acinetobacter baumannii,Stenotrophomonas maltophilia, Escherichia coli, Legionella.

Accordingly, in one embodiment the nosocomical infection is an infectioncaused from at least one bacterium as defined above in the section“Polyelectrolyte coating” herein above.

As further specified in the section “device” bacterial infection in formof a biofilm differ from conventional infections.

Accordingly, in one embodiment, the bacterial infection is a biofilminfection.

A “biofilm infection” herein refers to a bacterial infection withbiofilm forming bacteria. Biofilm forming bacteria are, for example, butis not limited to, gram-positive bacteria such as Staphylococcus, andgram-negative species such as Escherichia coli, or Pseudomonasaeruginosa.

Any combination of the above embodiments makes part of the invention.

Throughout the instant application, the term “comprising” is to beinterpreted as encompassing all specifically mentioned features as welloptional, additional, unspecified ones. As used herein, the use of theterm “comprising” also discloses the embodiment wherein no featuresother than the specifically mentioned features are present (i.e.“consisting of”). Furthermore the indefinite article “a” or “an” doesnot exclude a plurality. The mere fact that certain measures are recitedin mutually different dependent claims does not indicate that acombination of these measures cannot be used to advantage.

The invention will now be described in more detail with reference to thefollowing examples. All literature and patent documents cited herein arehereby incorporated by reference. While the invention has beenillustrated and described in detail in the foregoing description, theexamples are to be considered illustrative or exemplary and notrestrictive.

EXAMPLES 1. Example 1

1.1 Material

Polyelectrolyte multilayer coatings have been built up with thefollowing polyelectrolytes. Polycations of poly(I-argininehydrochloride) (PAR) were purchased from Alamanda Polymers, USA.Different PAR polymers used differ from the numbers of arginine perchain: PAR10 10 arginine (R), Mw=2.1 kDa, PDI=1); PAR30 (30 R, Mw=6.4kDa, PDI, =1.01), PAR100 (100 R, Mw=20.6 kDa, PDI=1.05), and PAR200 (200R, Mw=40.8 kDa, PDI=1.06). Poly(L-ornithine hydrochloride) (PLO) waspurchased from Alamanda Polymers, USA. Different PLO polymers useddiffer from the numbers of ornithine per chain: PLO30 (30 R, Mw=5.9 kDa,PDI, =1.03), PLO100 (100 R, Mw=18.5 kDa, PDI=1.03), and PLO250 (250 R,Mw=44.7 kDa, PDI=1.02). Poly(L-lysine hydrochloride) (PLL) such as PLL30(30 R, Mw=5.4 kDa, PDI, =1.02) was purchased from Alamanda Polymers,USA.

Hyaluronic acid (HA, Mw=150 kDa) used as the polyanion was from LifecoreBiomed, USA.

1.2 Methods

Monitoring Build-Up of Multilayer Coatings:

Coating or film build-up was followed using an in situ quartz crystalmicrobalance (QCM-D, E1, Q-Sense, Sweden). The quartz crystal is excitedat its fundamental frequency (about 5 MHz), as well as at the third,fifth, and seventh overtones (denoted by v=3, v=5, v=7 correspondingrespectively to 15, 25, and 35 MHz). Changes in the resonancefrequencies (Δf) are measured at these four frequencies. An increase ofΔf/v is usually associated to an increase of the mass coupled with thequartz. PAR (i.e. PAR10, PAR30, PAR50, PAR100 or PAR200) and HA weredissolved at 0.5 mg·mL⁻¹ in sterilized buffer containing 150 mM NaCl and10 mM of tris(hydroxymethyl)-aminomethan (TRIS, Merck, Germany) at pH7.4. The polyelectrolyte solutions were successively injected into theQCM cell containing the quartz crystal and PAR was the first depositedpolyelectrolyte. They were adsorbed for 8 min and then, a rinsing stepof 5 min with NaCl-Tris buffer was performed.

Buildup of (PAR/HA)₂₄ with Dipping Robot:

For the construction of 24 bilayers of PAR/HA ((PAR30/HA)₂₄) anautomated dipping robot was used (Riegler & Kirstein GmbH, Berlin,Germany). Glass slides (12 mm in diameter) were first washed withHellmanex® II solution at 2%, H₂O, and ethanol and dried with air flow.The solutions of polyelectrolytes were prepared as described above forQCM experiments. Glass slides were dipped alternatively in polycationand polyanion solutions and extensively rinsed in NaCl-Tris bufferbetween each step. After construction, the coatings were dried with airflow and then immerged in NaCl-Tris buffer and stored at 4° C. beforeuse. Thicknesses of obtained coatings were evaluated by deposition of100 μL of PLL-FITC (poly-L-lysine labelled with fluoresceinisothyocyanate, a green fluorescent probe) (0.5 mg·mL⁻¹ in Tris-NaClbuffer) on top of the PAR/HA multilayer coatings. After 5 minutes anddiffusion of PLL-FITC through the whole coating, a rinsing step wasperformed with Tris-NaCl buffer. Observations of the coatings werecarried out with a confocal microscope Zeiss LSM 710 microscope(Heidelberg, Germany) using a 20× objective (Zeiss, Plan Apochromat). 24bilayers of PLL/HA (PLL30/HA)₂₄ and 24 bilayers of PLO/HA (i.e.(PLO30/HA)₂₄, (PLO100/HA)₂₄ and (PLO250/HA)₂₄) were prepared in analogy,wherein PLO or PLL was dissolved at 0.5 mg·mL⁻¹ in sterilized buffercontaining 150 mM NaCl and 10 mM of tris(hydroxymethyl)-aminomethan(TRIS, Merck, Germany) at pH 7.4.

Antibacterial Assays:

Staphylococcus aureus (S. aureus, ATCC 25923) strains were used toassess the antibacterial properties of the test samples. Bacterialstrain was cultured aerobically at 37° C. in a Mueller Hinton Broth(MHB) medium (Merck, Germany), pH 7.4. One colony was transferred to 10mL of MHB medium and incubated at 37° C. for 20 h, to provide a finaldensity of 10⁶ CFU·mL⁻¹. To obtain bacteria in the mid logarithmic phaseof growth, the absorbance at 620 nm of overnight culture was adjusted of0.001.

Glass slides coated with (PAR/HA)₂₄ with PAR10, PAR30, PAR100, aresterilized by using UV-light during 15 minutes, then washed withNaCl-Tris buffer. After washing, each glass slides were deposited in24-well plates with 300 μl of S. aureus, A₆₂₀=0.001, and incubatedduring 24 hours at 37° C.

For negative control, uncoated glass slides were directly incubated withS. aureus using a similar method.

For positive control, Tetracycline (10 μg·mL⁻¹) and Cefotaxime (0.1μg·mL⁻¹) were added in S. aureus solution in contact with uncoated glassslides.

To quantify bacteria growth or inhibition after 24 h, the absorbance ofthe supernatant at 620 nm was measured.

The assay was performed similarly for Glass slides coated with(PLL30/HA)₂₄, (PLO30/HA)₂₄, (PLO100/HA)₂₄ and (PLO250/HA)₂₄.

The antibacterial assay for M. Luteus, E. Coli and P. aeruginosa wereperformed in analogy to the bacterial assay described for Staphylococcusaureus described above.

Bacteria Live Dead Assay:

To evaluate the health of bacteria which are on the surface, theBacLight™ RedoxSensor™ CTC Vitality Kit (ThermoFischer Scientific Inc.,France) was used. This kit provides effective reagents for evaluatingbacterial health and vitality. The kit contains 5-cyano-2,3-ditolyltetrazolium chloride (CTC), which has been used to evaluate therespiratory activity of S. aureus. Indeed, healthy bacteria will absorband reduce CTC into an insoluble, red fluorescent formazan product.Bacteria which are dead or with a slow respiratory activity will notreduce CTC and consequently will not produce red fluorescent product.Finally this kit gives a semi-quantitative estimate of healthy vsunhealthy bacteria. SYTO® 24 green-fluorescent nucleic acid stain(ThermoFischer Scientific Inc., France) is used for counting allbacteria. A solution of 50 mM CTC and 0.001 mM Syto 24 in pure water isprepared. Each glass slides were washed with phosphate-buffered salinebuffer, pH=7.4 (PBS) then 270 μl of PBS and 30 μL of CTC/Syto 24solution were added. The plate were incubated 30 minutes at 37° C., awayfrom light. Each surfaces were observed with confocal microscopy (ZeissLSM 710 microscope, Heidelberg, Germany), using a 63× objective,immerged in oil. Excitation/Emission wavelength of stains was 450/630 nmfor CTC and 490/515 nm for Syto 24.

Cytotoxicity Test:

Human fibroblast (CRL-2522 from ATCC/LGC Standards, France) was culturedat 37° C. in Eagle's Minimum Essential Medium (EMEM, ACC/LGC) with 10%of Fetal Bovin Serum (FBS, Gibco/ThermoFicher Scientific Inc., France)and 1% of penicillin streptomycin (Pen Strep, LifeTechnologies/ThermoFicher Scientific Inc., France). 50 000 cells wereincubated in each well of a 24 well-plates during 24 h. Glass slidescoated with (PAR/HA)₂₄ were incubated simultaneously in a 6 well-plateswith 1 mL of medium. After 24 h, the medium of the wells containingcells was removed and replaced by the supernatant that was in contactwith the multilayers for 24 h. Human fibroblasts were incubated during24 h at 37° C. Then, the supernatant was removed and incubated with 10%of AlamarBlue (ThermoFischer Scientific Inc., France) during 2 h. Thecell viability was determined by measuring the fluorescence of producedresofurin (Excitation/Emission wavelength=560/590 nm). Cells were washedtwice with PBS and fixed with PFA 4% solution during 10 minutes, andthen again washed twice with PBS. A solution of Phalloidin was preparedin PBS buffer with 1% of bovin serum albumin (BSA). The stainingsolution were placed on the fixed cells for 30 minutes at roomtemperature and washed two times with PBS buffer. A solution of DAPI wasprepared and placed on the cells at the same conditions as previously.Fluorescence images were captured using Nikon Elipse Ti-S with 63×PL APO(1.4 NA) objective equipped with Nikon Digital Camera (DS-Q11MC withNIS-Elements software, Nikon, France), and processed with ImageJ(http://rsb.info.nih.gov/ij/). Excitation/Emission wavelength forRhodamine Phalloidin was 540/565 nm and for DAPI 350/470 nm.

Time-Lapse Microscopy:

Glass slides coated with (PAR/HA)₂₄ were sterilized by using UV-lightduring 15 minutes, then washed with NaCl-Tris buffer. After washing,each glass slides were mounted in a Ludin Chamber (Life ImagingServices, Switzerland) at 37° C., 5% CO₂, with 1 mL μl of S. aureus(used as described previously, with A₆₂₀=0.001), stained with Syto 24during the culture. The time-lapse sequence was performed during 24 hwith a Nikon TIE microscope equipped with a 60×PL Apo oil (1.4 NA)objective and an Andor Zyla sCMOS camera (Andor Technology LtD. UnitedKingdom), was used with Nikon NIS-Elements Ar software (Nikon, France).Phase contrast and fluorescence images were acquired every 5 min for 24h. Images were processed with ImageJ.

Circular Dichroism:

Circular dichroism (CD) spectra were recorded using a Jasco J-810spectropolarimeter (Jasco Corporation, UK) as an average of 3 scansobtained using a 0.1 mm path length quartz cuvette at 22° C. from 180 to300 nm with data pitch of 0.1 nm and a scan speed of 20 nm/min. Allspectra were corrected by subtraction of the buffer spectra. Spectra foreach PAR were obtained at a concentration of 2 mg·mL⁻¹ in NaCl-TrisBuffer. All CD data were expressed as mean residue ellipticity.

Fluorescent Labelling of PAR:

For labeling PAR chains, PAR (15 mg·mL⁻¹ in 100 mM NaHCO₃ pH 8.3 buffer)was incubated with fluorescein isothiocyanate (FITC, Sigma Aldrich,France) at 1:2 molar ratio of PAR/FITC at room temperature for 3 h. Thissolution was dialyzed against 1 L of water at 4° C. with a Slide-A-LyserDialysis Cassette (Thermo Fischer Scientific Inc, USA), cut-off=3500MWCO. PAR-FITC was then produced and stored in aliquots of 2 mL (0.5mg·mL⁻¹ in NaCl-Tris buffer).

Release Experiments:

For the first experiment, a multilayer coating (PAR30/HA)₂₄ was built byusing PAR-FITC. Release experiments were performed at 37° C. during 24 hin presence of MHB medium or a S. aureus/MHB solution (A₆₂₀=0.001). 300μL of mineral oil were added on the top of the supernatant to preventany evaporation during the monitoring. The release of PAR-FITC insolution was performed by measuring the fluorescence of the supernatantover time with a spectrofluorimeter (SAFAS Genius XC spectrofluorimeter,Monaco) with excitation/emission wavelength of 488/517 nm. Three sampleswere studied for each conditions.

For the second experiment, a multilayer film (PAR30/HA)24 was incubatedat 37° C. with two conditions: A) with 300 μl of S. aureus solutionA620=0.001 and B) 300 μl of MHB only. After 24 h, the supernatant incontact with the LbL was taken and incubated with a new S. aureussolution to have a final A620=0.001. After 24 h at 37° C., theabsorbance at 620 nm was measured. Three samples were studied for eachcondition.

Fluorescence Recovery after Photobleaching (FRAP) Experiments:

The diffusion coefficient, D, and the proportion of mobile molecules, p,was measured for (PAR/HA)₂₄ multilayers containing PAR-FITC byperforming photobleaching experiments (FRAP, Fluorescence Recovery AfterPhotobleaching).

A glass slide coated with the multilayer was introduced in a home-madesample holder and immerged in 200 μl of Tris-NaCl buffer. One circularregions (4.4 μm in radius and referred as “R4” in an image of 35 μm×35μm or 10.6 μm in radius and referred as “R10” in an image of 85 μm×85μm) were exposed for 700 msec to the laser light set at its maximumpower (λ=488 nm). Then, the recovery of the fluorescence in the bleachedarea was observed over time. Observations were carried out with a ZeissLSM 710 microscope (Heidelberg, Germany) using a 20× objective (Zeiss,Plan Apochromat).

Cross-Linking of (PAR30/HA)24:

Crosslinking was performed by immersing the (PAR30/HA)24 films in asolution containing EDC (100 mM) and N-hydroxysuccinimide (10 mM) inNaCl (0.15 M) during 15 h at 4° C. Films were rinsed 2 times with a NaCl(0.15 M) solution. The films were immerged in a solution of ethanolamine(1M) during 40 minutes at 4° C. to neutralize all carboxylates functionsthat have not react. The films were rinsed with NaCl solution and theNaCl-Tris buffer solution was used for the last rinsing step.

1.3 Results

In order to test the buildup of the PAR/HA coatings, quartz crystalmicrobalance (QCM) was used. FIG. 1 corresponds to the layer-by-layerdeposition monitored with QCM for various molecular weight of PAR (10,30, 100 or 200 residues corresponding to notation PAR10 PAR30, PAR100 orPAR200 respectively). In a first approximation, it is known that theincrease in the normalized frequency could be related to an increase inthe deposited mass or thickness (REF). An exponential growth of thenormalized frequency with the number of deposition step was observed forcoatings buildup with PAR30, PAR100 or PAR200. The most important growthwas monitored for larger PAR chains. In the case of PAR₁₀ the incrementin the normalized frequency with the deposition number is the weaker,however an exponential growth was already observed (FIG. 1). Finally,despite the short length of this polypeptide, the layer-by-layer growthwas effective.

An opposite behavior was previously demonstrated for multilayer coatingsbuildup with chitosan/HA with various MW of chitosan (Richert 2004,Langmuir). An exponential growth of the coatings was observed for allthe MW of chitosan used, however the coating buildup was more rapid whenthe mass of the chitosan chains was smaller. This behavior was relatedto the diffusive properties of the chitosan chains in the coatings:shorter chains should diffuse more through the coating which should leadto a higher increase in the mass increment after each layer deposition.However in the present study, experimental conditions are different asan homopolypeptide was selected as the polycation instead of apolysaccharide and the range of their chain length was smaller.

Then, coatings with 24 bilayers ((PAR/HA)₂₄) were observed with confocalmicroscopy. In order to label fluorescently the coatings,poly(lysine)-FITC was added as the last layer on top of the coatings.Cross-section images of coatings build up with PAR of different residuenumbers depict thick bands with a green labeling through the wholecoating section (FIG. 2). This indicates that the coatings produce werehomogenously deposited on the surface in all conditions (for PAR with 10to 200 residues).

Next, the antimicrobial properties of (PAR/HA)24 multilayers for PAR ofdifferent number of residues was evaluated. The films were testedagainst a gram positive bacteria, S. aureus, a strain well known to beassociated with nosocomial infections and more particularly withimplant-related infections. For example in the case of orthopaedicimplants, S. aureus with S. epidermis is involved in 70% of infections(biomaterials 84, 2016, 301). S. aureus were incubated for 24 h at 37°C. in the presence of MHB medium on the (PAR/HA)24 coatings. Thebacteria were incubated at high density on surfaces for 24 h at 37° C.in the presence of MHB medium. The normalized growth of pathogens (%)was estimated by comparing absorbance at 620 nm in the presence ofmultilayer films in comparison with the positive control (withoutmultilayer films and in presence of antibiotics in the medium) and thenegative control (without multilayer films and in the absence ofantibiotics in the medium). No significant inhibition was observed forfilms built with PAR10, PAR50, PAR100 and PAR200. However, for PAR30 (30residues), more than 95% of bacterial growth inhibition was observedafter 24 hours. This suggests that PAR30 strongly impact viability of S.aureus. It must be pointed out that the molecular weight effect isextremely striking and up to now such an effect on the multilayer filmfunctionality, whatever this function, was never observed (see FIG. 3).

To evaluate more precisely the health of bacteria in contact with thesurfaces, the respiratory activity of S. aureus using5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was monitored. Healthybacteria will absorb and reduce CTC into an insoluble, red fluorescentformazan product and bacteria which are dead will not reduce CTC andconsequently will not produce fluorescent product (Data not shown). Atotal inhibition of bacteria on (PAR30/HA) surfaces was clearly observedand it was extremely rare to find an area with few bacteria (Data notshown). Comparatively, PAR10, PAR100 or PAR200 surfaces did not preventbacterial adhesion and growth, a similar density of healthy bacteria ason non-treated surfaces was found. This outstanding result is in fullcorrelation with growth inhibition in supernatant described above wherePAR30 was also the only coating strongly effective against bacteria.

In order to elucidate if the bacterial growth inhibition of the(PAR/HA)₂₄ coatings is only limited to S. aureus, bacterial growthinhibition of (PAR30/HA)₂₄ was further tested against other grampositive and gram negative bacteria. Accordingly, the antimicrobialproperties of (PAR30/HA)₂₄ multilayer was evaluated for methilinresistant S. aureus strain, M. Luteus, E. coli and P. aeruginosa. Theresult shown in FIG. 16 demonstrates that the coating has anantibacterial activity against different gram negative and gram positivebacteria.

In this context, the inventors were interested if the antibacterialactivity is limited to the herein described (PAR/HA) multilayer coatingsor if a coating comprising as polycation another polypeptide would alsodemonstrate the same antibacterial activity. The inventors of thepresent invention further evaluated (PLL/HA)₂₄ and (PLO/HA)₂₄multilayers for PLL and PLO of different number of residues.Surprisingly, as it can be concluded from FIGS. 4, 6, 9, 10 and 17 also(PLL/HA)₂₄ and (PLO/HA)₂₄ multilayers showed high growth inhibition forA. aureus and M. Luteus.

In the following the inventors of the present invention wanted tofurther elucidate the underlying mechanism that confer to the coatingsof the invention the strong inhibitory property on the surface and inthe supernatant. As an example the (PAR30/HA)₂₄ coating was furtherinvestigated. Accordingly, the minimal inhibitory concentration (MIC) ofPAR in solution was determined using bacterial assay as described inExperimental Section. For concentrations up to 0.04 mg·mL⁻¹, all PAR(PAR10, PAR30, PAR100 or PAR200) totally inhibited S. aureus growth(FIG. 11). However when PAR concentrations were decreased, a discrepancybetween PAR was observed: a total inhibition of S. aureus growth wasmonitored for all PAR at concentrations of 0.03 mg·mL⁻¹ except for thelonger one, i.e. PAR200 where only a partial inhibition (about 45%) wasmeasured. Finally for PAR at concentrations of 0.01 mg·mL⁻¹, inhibitionof 100% was shown only for PAR₃₀. Longer or shorter PAR chains (PAR10,PAR100 or PAR200) inhibits only partially (less than 40%) S. aureusgrowth. This suggests that PAR30 is more effective in solution. Thisreasoning is valid when PAR concentration values are expressed in mass(mg·mL⁻¹) thus it is related to the number of arginine monomers. Howeverwhen the graph is plotted with concentrations in μM (and thusproportional in number of chains), which means that concentration isrelated to number of chains, different interpretation can be made. Atlow concentrations, longer chains are more effective: at 1 μM, PAR100and PAR200 totally inhibit bacterial growth whereas for a similar effectPAR30 needs to be at about 2 μM. Finally, the inventors of concludedfrom these results that all PAR chains are effective in solution toinhibit S. aureus growth. For a given mass of PAR, PAR30 is the mosteffective. Moreover the MIC₁₀₀ values obtained for PAR30, PAR100 orPAR200 ranges at very low concentrations (between 1 and 2 μM) which isremarkable when compared to well-known antimicrobial peptides (forexample 30 μM for cateslytin with S. aureus, see Adv. Funct. Mater.2013, 23, 4801-4809). PAR is thus a powerful candidate to fight againstS. aureus. However, even if there are differences in the MICs of the PARof different length, they do not differ by orders of magnitude and thusseem not to be, at least solely, at the origin of the striking molecularweight effect observed on PAR/HA multilayers.

To address the conformations of PAR chains and to check if PAR30 chainshave a specific secondary structure that could explain their inhibitoryproperties compare to longer or shorter chains, circular dichroism (CD)experiments were performed. Firstly, secondary structures of PAR chainsin NaCl/TRIS buffer solution (150 mM NaCl, 10 mM Tris, pH 7.4) (Data notshown). All CD spectra of PAR chains (PAR10, PAR30, PAR100 and PAR200)show a unique negative minimum at about 200 nm characteristic of arandom coil conformation in solution. In a second step, PAR conformationin PAR/HA multilayer coatings was monitored (Data not shown).Surprisingly, spectra of the coatings depict totally different profiles:no more minima at 200 nm were observed, however one minima at about 10and another one at about 222 nm were monitored, except for (PAR10/HA)24,which present a unique negative minimum at 200 nm (random coil). Theycan probably not be attributed to HA chains as it is known that insolution at pH 7.4 they have an unordered conformation (Zahouani, ACSApplied Materials, 2016, in press). Moreover the PAR/HA spectrumcorrespond more probably to chains in α-helix conformationscharacterized by these two minimums. This indicates that PAR chainsshould change from a coil conformation in solution to an α-helix in thecoating. Similar behavior were previously observed for LbL build up withpolylysine and poly(glutamic acid) (Boulmedais F. et al. Langmuir, 2002,18, 4523). Interestingly, unordered antimicrobial peptides are known toadopt an α-helix conformations when they interact with the bacterialmembrane and this mechanism is a key point in their mechanism of action(Porcellini F. et al. 2008, Biochemistry, 47, 5565 and Lugardon K. etal., 2001, J Biol Chem., 276(38): 35875). Here in polyelectrolytemultilayer coatings, PAR chains already adopt an α-helix conformationmost probably due to the interactions between PAR and HA and local highconcentration of PAR. This mechanism can be helpful to fight faster andin a more efficiency way against invading bacteria.

But, because the films built with different PAR chain lengths presentsimilar spectra, the secondary structure of PAR chains cannot explainthe striking molecular weight effect on the bactericidal property of thePAR30/HA films.

In view of the absence of specific properties of PAR30 in solutioncompared to shorter or longer PAR chains, the antimicrobial abilities ofPAR30/HA films should be related to the film property by itself. In thiscontext, we investigated if the bactericidal property of the film is dueto the release of PAR30 chains from the multilayer into the solution orif bacteria need to come in contact with the film to be killed. For thispurpose two types of experiments were performed. Using fluorescentlylabelled PAR30 chains we first determined the release of PAR30 chainsinto the solution containing solely MH medium with and without S.aureus. FIG. 13 shows a typical release kinetics curve. Indeed, a slowrelease process over a time scale of the order of 24 h was observed butit clearly comes out that even after 24 h the PAR30 concentrationreached in solution lies significantly below the corresponding MICconcentration: PAR30 released is about 0.18 μM after 24 h while MIC90 isabout 2 μM. Moreover, when the supernatant, after 24 h of contact withthe film, was brought in contact with suspension containing bacteria ata final concentration identical to previous experiments, absolutely nobacteria growth inhibition was observed, confirming that the MIC was notreached in supernatant (FIG. 15). Finally, we also performed anexperiment where bacteria were brought in contact with a (PAR30/HA)24film for 24 hours. Bacteria growth was totally inhibited. Thesupernatant of this experiment was removed and brought it in contactwith a fresh suspension of bacteria. Here again, the bacteria growth wasno further inhibited (Data not shown). These results demonstrate thatthe release of the PAR30 chains from the film in the supernatant cannotbe at the origin of the bactericidal effect. Finally we can hypothesizethat the bactericidal effect is directly related to the contact of thebacteria with the PAR30/HA film which acts as a contact-killingmultilayer.

Then the inventors investigated if the bactericidal activity of thePAR30/HA multilayer is related to the mobility of these chains in thefilms. Indeed, it is known that the exponential character of amultilayer is related to the diffusion ability of at least one of itsconstituents in and out of the film during each deposition step. Theyfirst determined the mobility of the different chains, PAR10, PAR30,PAR100 and PAR200 in the (PAR/HA)24 multilayer by using FRAP method.FIG. 14 shows the evolution of the normalized fluorescence in thebleached area as a function of the square root of time. From thesecurves the percentage of immobile chains over the timescale of theexperiments can be deduced. It clearly appears that more than 80% of thePAR10 and PAR30 chains are mobile and that only 60% and 20% of PAR100and PAR200 respectively are mobile (Data not shown). The fraction ofmobile chains dramatically decreases when the chain length increasesfrom 30 to 100 or 200 residues.

The inventors also cross-linked the PAR30/HA multilayers using astandard EDC-NHS cross-linking method between amine and carboxylicgroups. After cross-linking we found that the proportion of immobilechains measured by FRAP increases (Data not shown). When such a film wasbrought in contact with S. aureus, only 30% inhibition of the bacterialgrowth was observed after 24 hours of contact. These results suggestthat the percentage of mobile PAR chains in the multilayer is animportant parameter controlling its bactericidal property of the film(FIG. 11).

Finally, using confocal microscopy, the inventors also investigated thestructure of the film after 24 hours of contact with bacteria (Data notshown). For this purpose, films were constructed by incorporatingfluorescently labelled PAR30 chains. It was found that after 24 hours ofcontact, the film is no longer homogeneous but that non-fluorescentareas appear. Because these areas have a smooth shape, they suggest areorganization of the film. Such a behavior is not observed in theabsence of bacteria where the films remain homogeneous. Such areorganization could be consecutive to de decrease of the PAR chains inthe film and suggest the following bactericidal mechanism: When bacteriacome in contact with the multilayer, the negative bacteria membranes actas strong attractive surfaces for the PAR chains. Mobile PAR chains arethus soaked out of the film by the bacteria membranes and destabilizethem. Chains of large molecular weight have a stronger membranedestabilization power than smaller molecular weight ones as it comes outfrom the MICs determined in solution. PAR10 chains are 10 times lessactive than PAR100 or PAR200 chains but PAR30 chains are only 2 timesless active than PAR100 or PAR200 chains. Yet, in a film one can assumethat the concentration of arginine monomers is fairly independent of themolecular weight of the PAR chains. Thus, the concentration of chainsdecreases when the molecular weight of the polyelectrolytes increases.For example the concentration PAR30 chains in the film should be 3 timeshigher than that of PAR100. In addition there are of the order of 90% ofPAR10 chains that are mobile whereas only 70% of PAR100 chains aremobile. This leads to 4 times more PAR10 chains than PAR100 in the film.There could be other factors explaining the higher propensity of filmsPAR/HA films built with PAR30 to be strongly bactericidal but the chainmobility is without doubt an important one.

To summarize, the inventors of the present invention have found thatmultilayer coatings comprising PAR, PLL or PLO as polycation and HA aspolyanion present a strong anti-microbial effect against S. aureus, M.Luteus, E. coli and P. aeruginosa. This effect strikingly depends on themolecular weight of the polypeptide chains. This effect is explained bythe concentration of mobile in the multilayers and their power to killbacteria as a function of the molecular weight. These results open theroute to new type of applications of polyelectrolyte multilayers, inparticular of antibacterial multilayers, where the function can be tunedby the molecular weight of the polyelectrolytes.

2. Example 2

2.1 Material

Polyelectrolyte multilayer coatings have been built up using thepolyelectrolytes described herein above in section 1.1. The followingpolyelectrolytes were used in addition: Polycations of poly(I-argininehydrochloride) (PAR) were purchased from Alamanda Polymers, USA.Different PAR polymers used differ from the numbers of arginine perchain: PAR50 (50 arginine (R), Mw=9.6 kDa, PDI=1.03); PAR70 (70 arginine(R), Mw=13.4 kDa, PDI, =1.01), PAR150 (150 arginine (R), Mw=29 kDa,PDI=1.04).

2.2 Methods

The methods used are as described herein above in the correspondingsection under paragraph 1.2.

2.3 Results

2.3.1 Effect of Number of Arginine Residues on PAR/HA AntimicrobialActivity: Coatings with PAR30, PAR50, PAR70, PAR100 and PAR150 after 24Hours of Incubation

PAR with 30, 50, 100 and 150 residues have been tested in order toconfirm previous results and to obtain complimentary results.Measurements were performed with a glass slide coated with (PAR/HA)₂₄(i.e. 24 layers of PAR alternating with 24 layers of HA) and placed in a24 well-plate, as previously described (Chem. Mater. 2016, 28, 8700).300 μL of S. aureus at a concentration of 8.105 CFU·mL⁻¹ was depositedin each well and incubated for 24 h at 37° C. Then the absorbance of thesupernatant at 620 nm was measured.

PAR50/HA Versus PAR30/HA with 24 Bilayers

PAR50/HA films built up with 24 bilayers show a complete bactericideeffect on bacteria (FIGS. 19 and 20). Moreover, observations withconfocal microscope using CTC/Syto24 labeling show mainly no bacteria onthe coatings (data not shown).

PAR50/HA Versus PAR30/HA with 48 Bilayers

Increasing the number of bilayer from 24 to 48 show similar results:inhibition of S. aureus growth is total, for coatings based either onPAR50 or on PAR30 (FIGS. 21 and 22). These results were confirmed withobservations with confocal microscopy: no bacteria were observed onPAR50/HA coatings or PAR30/HA coatings.

PAR100/HA and PAR150/HA Versus PAR30/HA with 24 Bilayers

PAR100/HA coatings built with 24 bilayers depict a total antimicrobialactivity (FIG. 23). On the other hand, PAR150/HA coatings did notinhibit bacteria, the coating is not effective at all. Confocalmicroscopy observations confirm these results, no bacteria were observedon PAR100/HA, and PAR30/HA coatings but many bacteria could bevisualized on PAR150/HA coatings.

PAR10/HA Versus PAR30/HA with 24 Bilayers

PAR10/HA coatings with 24 bilayers totally inhibit bacteria in thesupernatant (FIG. 24). As controls, PAR50/HA and PAR30/HA coatingsconfirm their antimicrobial activity as described above. Similar resultscould be drawn from observation of surfaces with confocal microscopy.

PAR70/HA Versus PAR30/HA with 24 Bilayers

Bacteria in the supernatant where a PAR70/HA coating is placed aretotally inhibited (FIG. 25). This was confirmed by confocal experiments.

2.3.2. Long Term Antimicrobial Activity of PAR/HA Coatings: PAR10,PAR30, PAR50, PAR100 and POR30 after 24/48 or 72 h of Incubation

PAR30/HA versus POR30/HA with 24 bilayers after 24/48 or 72 h ofincubation

No bacteria were monitored after 24, 48 or 72 h in the supernatant when(PAR30/HA)₂₄ or (POR30/HA)₂₄ coatings were used (FIGS. 26 to 28).Similar conclusions can be drawn when surfaces are visualized withconfocal microscope.

PAR10/HA, PAR50/HA and PAR100/HA with 24 Bilayers after 24/48 or 72 h ofIncubation

Similar experiments were performed with PAR10/HA, PAR50/HA and PAR100/HAcoatings. After 24 h, no bacteria were measured (FIG. 29) in thesupernatant for both coatings. PAR50/HA or PAR100/HA coatings at 48 h,PAR10/HA coatings is no more effective, S. aureus growth is at a levelcomparable to surfaces without coatings (FIG. 30). Similar results areobtained at 72 h where bacteria are alive with a PAR10/HA coating butare dead with PAR50/HA or PAR100/HA coatings (FIG. 31). All theseresults were confirmed with confocal microscope observations.

2.3.3 Storage and Sterilization of PAR/HA Coatings

Storage of PAR/HA Coatings

In order to check if drying procedures and storage of PAR/HA coatingsallow to maintain or not the antimicrobial activity, a (PAR30/HA)24coating was tested after drying it (rinsing with pure water and dryingat ambient temperature) and storage at 4° C. for 1 or 7 days (FIGS. 32and 33). No change in the antimicrobial activity was observed afterthese two processes, absolutely no bacteria were able to growth in thesupernatant of the wells containing the coatings. This indicates thatfilms are probably stable after a drying procedure and storage forseveral days did not modify its properties.

Sterilization of PAR/HA Coatings

Activity of (PAR30/HA)₂₄ coatings have been tested after a dryingprocedure and an autoclave sterilization following regular cycles usedfor sterilization of medical devices (30 minutes with cycles at 121° C.)(FIG. 34). This sterilization protocol did not modify the antimicrobialactivity of the coating; no change in the total bactericide activity wasmeasured.

2.4. Conclusions

Finally several conclusions can be drawn from these studies:

(PAR/HA)₂₄ coatings built with PAR10, PAR30, PAR50, PAR70, PAR100 show atotal antimicrobial activity against S. aureus after 24 h ofinoculation. However, in our previous preliminary studies, PAR10 andPAR100 were not always active with 24 h. This is probably because PAR10and PAR100 correspond to chain lengths at the limit of values which areeffective. On the contrary, PAR30 and PAR50 always show a totalantimicrobial activity in our experiments (at least more than 10individual experiments for both have been realized). (PAR150/HA)₂₄coatings never show some antimicrobial activities after 24 h ofincubation of S. aureus.

(PAR/HA)₂₄ coatings built with PAR30, PAR50, PAR100 and POR30 after24/48 or 72 h of incubation show a total inhibition of bacteria whichdemonstrate their efficiency over 3 days and three successivecontaminations. On the contrary, PAR10 is no more active after the thirdinoculation (72 h).

(PAR/HA)24 coatings can be stored at 4° C. for several days after dryingwithout any loss in their activity. Moreover application of standardsterilization protocol used for medical devices can be apply to(PAR/HA)24 coatings, the antimicrobial properties of the coatingactivity will be maintained.

FIGURES

FIG. 1: Graph demonstrating the buildup of (PAR/HA) multilayer coatingon a SiO₂ coated crystal followed by QCM. Various molecular weight ofPAR (10, 30, 100 or 200 residues corresponding to notation PAR10 PAR30,PAR100 or PAR200 respectively) are used in association with HA.Evolution of the normalized frequency−Δfv/v (for v=3) as a function ofthe number of layers adsorbed. An exponential growth of the normalizedfrequency with the number of deposition step was observed for coatingsbuildup with PAR30, PAR100 or PAR200. The most important growth wasmonitored for larger PAR chains. In the case of PAR10 the increment inthe normalized frequency with the deposition number is the weaker,however an exponential growth was already observed.

FIG. 2: Image showing the confocal microscopy images of PAR/HA coatingsections (x,z). Observation by confocal microscopy of PAR/HA coatingsections (x,z) for PAR/HA coating buildups of (PAR/HA) multilayercoating on a SiO₂ coated crystal followed by QCM. The Buildup of(PAR/HA) multilayer coating with PAR of various molecular weight wascompared, i.e. PAR10 PAR30, PAR100 or PAR200. This images indicate thatthe obtained coatings were homogenously deposited on the surface in allconditions (for PAR with 10 to 200 residues).

FIG. 3: Graph demonstrating the growth inhibition of S. aureus using thePAR coatings of the invention. The normalized S. aureus growth (%)obtained in a supernatant after 24 h in contact with (PAR/HA)₂₄multilayer coatings composed of poly-L-arginine with various number ofresidues is shown. Each value corresponds to the mean value of 3experiments and error bars correspond to standard deviations. The growthof S. aureus is about 80% for (PAR10/HA)₂₄, 75% for (PAR100/HA)₂₄, about90% for (PAR200/HA)₂₄ and less than 5% for (PAR30/HA)₂₄ thus showing astrong growth inhibition of more than 95% for (PAR30/HA)_(24.)

FIG. 4: Graph demonstrating the growth inhibition of S. aureus using thePLL coatings of the invention. The normalized S. aureus growth (%)obtained in a supernatant after 24 h (J+1), after 2 d and after 3 daysin contact with (PLL30/HA)₂₄. multilayer coatings is shown. The coatingwas put in contact with a fresh suspension of S. aureus after each 24 h.Each value corresponds to the mean value of 3 experiments and error barscorrespond to standard deviations. The growth of S. aureus is less than5% for (PLL30/HA)₂₄ after 1 and 2 days, thus showing a strong growthinhibition of more than 95% for (PLL30/HA)₂₄ in the first 48 hrs.

FIG. 5: Graph demonstrating the growth inhibition of M. luteus using thecoating (PAR30/HA)₂₄. The normalized M. luteus growth (%) observed in asupernatant after 20 h in contact with (PAR30/HA)₂₄ multilayer coatingsis shown. Each value corresponds to the mean value of 3 experiments anderror bars correspond to standard deviations. The growth of M. luteus isless than 2% for (PAR30/HA)₂₄ thus showing a strong growth inhibition ofmore than 98% for (PAR30/HA)_(24.)

FIG. 6: Graph demonstrating the growth inhibition of S. aureus usingPAR30 or PLL30 coatings in the absence of the polyanion HA. Thenormalized S. aureus growth (%) obtained in a supernatant after 24 h incontact with a polycationic layer of PLL30 or PAR30 in the absence of apolyanion layer HA is shown. The coating was put in contact with a freshsuspension of S. aureus for 24 h. Each value corresponds to the meanvalue of 3 experiments and error bars correspond to standard deviations.The growth of S. aureus is about 65% for PLL30 and about 100% for PAR30thus showing only a slight growth inhibition of 35% for PLL30 and nogrowth inhibition for PAR30.

FIG. 7: Graph demonstrating the growth inhibition of S. aureus using(PAR200/HA)₂₄-PAR30 of the invention. A final layer PAR30 was added onthe multilayer coating (PAR200/HA)₂₄ thus producing the multilayercoating PAR200/HA)₂₄-PAR30. The normalized S. aureus growth (%) obtainedin a supernatant after 24 hrs in contact with (PAR200/HA)₂₄-PAR30multilayer coatings is shown. Each value corresponds to the mean valueof 3 experiments and error bars correspond to standard deviations. Thegrowth of S. aureus is less than 1% for (PAR200/HA)₂₄-PAR30 thus showinga strong growth inhibition of more than 99% for (PAR200/HA)₂₄-PAR30

FIG. 8: Graph demonstrating the growth inhibition of M. Luteus using(PAR200/HA)₂₄-PAR30 of the invention. A final layer PAR30 was added onthe multilayer coating (PAR200/HA)₂₄ thus producing the multilayercoating (PAR₂₀₀/HA)₂₄-PAR₃₀. The normalized M. Luteus growth (%)observed in a supernatant after 24 h in contact with PAR200/HA)₂₄-PAR30multilayer coatings is shown. Each value corresponds to the mean valueof 3 experiments and error bars correspond to standard deviations. Thegrowth of M. luteus is less than 1% for (PAR200/HA)₂₄-PAR30 thus showinga strong growth inhibition of more than 99% for (PAR200/HA)₂₄-PAR30.

FIG. 9: Graph demonstrating the growth inhibition of S. aureus using thePLO coatings of the invention. The normalized S. aureus growth (%)obtained in a supernatant after 24 h in contact with (PLO/HA)₂₄multilayer coatings composed of poly-L-ornithine with different numberof residues is shown i.e. (PLO30/HA)₂₄ et (PLO100/HA)₂₄. Each valuecorresponds to the mean value of 3 experiments and error bars correspondto standard deviations. The growth of S. aureus is about 80% for(PLO250/HA)₂₄, less than 5% for (PLO100/HA)₂₄ and less than 3% for(PLO30/HA)₂₄ thus showing a strong growth inhibition of more than 95%for (PLO100/HA)₂₄ and (PLO30/HA)_(24.)

FIG. 10: Graph demonstrating the growth inhibition of S. aureus using(PLL₃₀/HA)₂₄ or crosslinked (PLL₃₀/HA)₂₄. (PLL30/HA)₂₄ was cross-linkedusing EDC/NHS with 0.5 M EDC and 0.1M NHS for 15 h at 4° C. Unreactedcarboxyl groups were neutralized using ethanolamine. The normalized S.aureus growth (%) was measured in a supernatant after 24 h in contactwith (PLL₃₀/HA)₂₄ or crosslinked (PLL₃₀/HA)₂₄ multilayer coatings. Thegrowth of S. aureus is about 65% for crosslinked (PLL₃₀/HA)₂₄ and lessthan 18% for (PLL30/HA)₂₄ thus showing that crosslinking significantlyreduces the biocidal activity of the coating.

FIG. 11: Graph demonstrating the growth inhibition of S. aureus using(PAR₃₀/HA)₂₄ or crosslinked (PAR₃₀/HA)₂₄. (PAR30/HA)₂₄ was cross-linkedusing EDC/NHS with 0.5 M EDC and 0.1M NHS for 15 h at 4° C. Unreactedcarboxyl groups were neutralized using ethanolamine. The normalized S.aureus growth (%) was measured in a supernatant after 24 h in contactwith (PAR₃₀/HA)₂₄ or crosslinked (PAR₃₀/HA)₂₄ multilayer coatings. Thegrowth of S. aureus is about 65% for crosslinked (PAR3₀/HA)₂₄ and lessthan 5% for (PLL30/HA)₂₄ thus showing that crosslinking reduces thebiocidal activity of the coating.

FIG. 12: Graph with the Minimal inhibitory concentration (MIC₁₀₀) ofsoluble PAR with 10, 30, 100 or 200 arginine residues. The Minimalinhibitory concentration (MIC₁₀₀) leading to 100% inhibition of S.aureus were measured in solution. PAR with 10, 30, 100 or 200 arginineresidues were tested. For concentrations up to 0.04 mg·mL⁻¹, all PAR(PAR₁₀, PAR₃₀, PAR₁₀₀ or PAR₂₀₀) totally inhibited S. aureus growth

FIG. 13: Graph showing the results of release experiments. Releaseexperiments were performed as described in the section “releaseexperiments” herein above. The multilayer coating (PAR30FITC/HA)₂₄ withPAR-FITC was then contacted with MHB medium or a S. aureus/MHB solution(A₆₂₀=0.001). The release of PAR-FITC was the monitored over the time.Three samples were studied for each condition.

FIG. 14: Graph demonstrating the proportion of mobile PAR (%) in(PAR/HA)24 coating according to MW. The diffusion coefficient, D, andthe proportion of mobile molecules, p, was measured for (PAR/HA)24multilayers containing PAR-FITC by performing photobleaching experiments(FRAP, Fluorescence Recovery After Photobleaching) as described above inthe section “Fluorescence Recovery After Photobleaching (FRAP)experiments”.

FIG. 15: Graph demonstrating that the concentration of PAR30 thatdiffuses from the coating into a solution is insufficient to efficientlyinhibit growth of S. aureus. Normalized Growth of S. aureus after 24 hin contact with a medium incubated with a multilayer film (PAR30/HA)₂₄.Medium A) with 300 μl of S. aureus solution A620=0.001 and B) 300 μl ofMHB only. No bacteria growth inhibition was observed.

FIG. 16: Graph demonstrating the growth inhibition of different bacteriausing (PAR₃₀/HA)₂₄ Normalized Growth of bacteria after 24 h in contactwith glass covered with the coating (PAR30/HA)₂₄. Bacteria growth isinhibited by more than 90% for S. aureus, methilicin resistant S.aureus, M. Luteus, E. Coli and P. aeruginosa.

FIG. 17: Graph demonstrating the growth inhibition of S. aureus usingthe PLL coatings of the invention. The normalized S. aureus growth (%)obtained in a supernatant after 24 h in contact with (PLL/HA)₂₄multilayer coatings composed of poly-L-lysine with different number ofresidues is shown i.e. (PLL10/HA)₂₄, (PLL30/HA)₂₄, (PLL100/HA)₂₄ and(PLO250/HA)₂₄. Each value corresponds to the mean value of 3 experimentsand error bars correspond to standard deviations. The growth of S.aureus is less than 3% for the combination of glass and antibiotics andless than 6% for (PLL30/HA)₂₄ whereas the bacterial growth is about 75%for (PLO250/HA)₂₄; about 85% for (PLL100/HA)₂₄ and about 90% for(PLL10/HA)_(24.)

FIG. 18: Graph demonstrating the growth inhibition of S. aureus overtime using (PAR30/HA)24. The normalized S. aureus growth (%) obtained ina supernatant after 1 day, 2 days and three days in contact with(PAR30/HA)₂₄ multilayer coating is shown. The coating was put in contactwith a fresh suspension of S. aureus after each 24 h. Each valuecorresponds to the mean value of 3 experiments and error bars correspondto standard deviations. The growth of S. aureus is less than 5% for(PAR30/HA)₂₄ after 1 and 2 days, thus showing a strong growth inhibitionof more than 95% for (PAR30/HA)₂₄ in the first 48 hrs. After the 3 daythe inhibitory activity is reduced.

FIGS. 19 and 20: Graph demonstrating the growth inhibition of S. aureususing the PAR coatings of the invention. Evaluation of antimicrobialactivities in the supernatant of (PAR5O/HA)₂₄ coatings and comparisonwith (PAR30/HA)₂₄ and controls (glass without coatings noted as “Glass”or “glass+antibiotics”). Each experiments is done with 3 glass slidesand a) and b) correspond to 2 similar independent experiments. “Medium”condition means wells without bacteria, only the OD of the medium ismeasured. Error bars correspond to standard deviations.

FIGS. 20 and 21: Graph demonstrating the growth inhibition of S. aureususing the PAR coatings of the invention. Evaluation of antimicrobialactivities in the supernatant of (PAR5O/HA)48 coatings and comparisonwith (PAR30/HA)48 and controls (glass without coatings noted as “Glass”or “glass+antibiotics”). Each experiments is done with 3 glass slidesand a) and b) correspond to 2 similar independent experiments. “Medium”condition means wells without bacteria, only the OD of the medium ismeasured. Error bars correspond to standard deviations.

FIG. 23: Graph demonstrating the growth inhibition of S. aureus usingthe PAR coatings of the invention. Evaluation of antimicrobialactivities in the supernatant of (PAR100/HA)₂₄ (PAR150/HA)₂₄ coatingsand comparison with (PAR30/HA)₂₄ and controls (glass without coatingsnoted as “Glass” or “glass+antibiotics”). Each experiment is done with 3glass slides. “Medium” condition means wells without bacteria, only theOD of the medium is measured. Error bars correspond to standarddeviations.

FIG. 24: Graph demonstrating the growth inhibition of S. aureus usingthe PAR coatings of the invention. Evaluation of antimicrobialactivities in the supernatant of (PAR10/HA)₂₄ coatings and comparisonwith (PAR50/HA)₂₄ and (PAR30/HA)₂₄ and controls (glass without coatingsnoted as “Glass” or “glass+antibiotics”). Each experiment is done with 3glass slides. “Medium” condition means wells without bacteria, only theOD of the medium is measured. Error bars correspond to standarddeviations.

FIG. 25: Graph demonstrating the growth inhibition of S. aureus usingthe PAR coatings of the invention. Evaluation of antimicrobialactivities in the supernatant of (PAR70/HA)₂₄ coatings and comparisonwith controls (glass without coatings noted as “Glass” or“glass+antibiotics”). Each experiment is done with 3 glass slides.“Medium” condition means wells without bacteria, only the OD of themedium is measured. Error bars correspond to standard deviations.

FIGS. 26, 27 and 28: Graph demonstrating the growth inhibition of S.aureus using the PAR coatings of the invention over time. Evaluation ofantimicrobial activities in the supernatant of (PAR30/HA)24 (POR30/HA)24coatings and comparison with controls (glass without coatings noted as“Glass” or “glass+antibiotics”) after incubation of S. aureus for 24, 48or 72 h. At t=0, 24 h and 48 h, a new inoculation with bacteria isperformed. Each experiment is done with 3 glass slides. “Medium”condition means wells without bacteria, only the OD of the medium ismeasured. Error bars correspond to standard deviations.

FIGS. 29, 30 and 31: Graph demonstrating the growth inhibition of S.aureus using the PAR coatings of the invention over time. Evaluation ofantimicrobial activities in the supernatant of (PAR10/HA)₂₄,(PAR50/HA)₂₄, (PAR100/HA)₂₄ coatings and comparison with controls (glasswithout coatings noted as “Glass” or “glass+antibiotics”) afterincubation of S. aureus for 24, 48 or 72 h. At t=0, 24 h and 48 h, a newinoculation with bacteria is performed. Each experiment is done with 3glass slides. “Medium” condition means wells without bacteria, only theOD of the medium is measured. Error bars correspond to standarddeviations.

FIGS. 32 and 33: Graph demonstrating the growth inhibition of S. aureusafter exposing the PAR coatings of the invention to different storageconditions. Evaluation of antimicrobial activities in the supernatant of(PAR30/HA)₂₄ coatings that was previously dried and stored at 4° C. for1 day (a) or 7 days and comparison with controls (glass without coatingsnoted as “Glass” or “glass+antibiotics”). Each experiment is done with 3glass slides. “Medium” condition means wells without bacteria, only theOD of the medium is measured. Error bars correspond to standarddeviations.

FIG. 34: Graph demonstrating the growth inhibition of S. aureus afterexposing the PAR coatings of the invention to different storageconditions. Evaluation of antimicrobial activities in the supernatant of(PAR30/HA)₂₄ coatings that was previously sterilized by autoclave andcomparison with controls (glass without coatings noted as “Glass” or“glass+antibiotics”). Each experiment is done with 3 glass slides.“Medium” condition means wells without bacteria, only the OD of themedium is measured. Error bars correspond to standard deviations.

The invention claimed is:
 1. A polyelectrolyte coating comprising: (a)from 18 to 60 polycationic layers consisting of at least one polycationconsisting of n repetitive units having the formula (1),

wherein n is an integer comprised between 11 and 85, and each R group,identical or different, is selected from the group consisting of —NH₂,—CH₂—NH₂ and —NH—C(NH)—NH₂, and (b) from 18 to 60 polyanionic layersconsisting of hyaluronic acid, wherein said polyelectrolyte coating hasantimicrobial activity.
 2. The polyelectrolyte coating according toclaim 1, wherein the polycationic layers consist of n repetitive unitshaving the formula (1),

wherein n is an integer comprised between 15 and 85, and R is selectedfrom the group consisting of —NH₂, —CH₂—NH₂ and —NH—C(NH)—NH₂.
 3. Thepolyelectrolyte coating according to claim 1, wherein R of formula (1)is —NH—C(NH)—NH₂.
 4. The polyelectrolyte coating according to claim 1,wherein n is 20 to
 85. 5. The polyelectrolyte coating according to claim1, which comprises 18 to 50 polycationic layers.
 6. The polyelectrolytecoating according to claim 1, which comprises 18 to 50 polyanioniclayers.
 7. A device having biocidal activity comprising apolyelectrolyte coating according to claim
 1. 8. The device of claim 7,wherein the polyelectrolyte coating covers at least a portion of thesurface of said device.
 9. The device of claim 8, wherein said device isan implantable device.
 10. The device according to claim 9, wherein theimplantable device is selected from the group comprising catheters,arteriovenous shunts, breast implants, cardiac and other monitors,cochlear implants, defibrillators, dental implants, maxillofacialimplants, middle ear implants, neurostimulators, orthopedic devices,pacemaker and leads, penile implants, prosthetic devices, replacementjoints, spinal implants, voice prosthesis, artificial hearts, contactlenses, fracture fixation device, infusion pumps, intracranial pressuredevice, intraocular lenses, intrauterine devices, joint prosthesis,mechanical heart valves, orthopedic devices, suture materials, urinarystents, vascular assist device, vascular grafts, vascular shunts andvascular stents, and artificial vessels of permanent or transient types.11. A method for preparing a device comprising the polyelectrolytecoating according to claim 1, the method comprising: (a) providing adevice; (b1) depositing on the surface of said device (i) 18 to 60polycationic layers consisting of at least one polycation consisting ofn repetitive units having the formula (1),

wherein n is an integer comprised between 11 and 85, and each R group,identical or different, is selected from the group consisting of —NH₂,—CH₂—NH₂ and —NH—C(NH)—NH₂, and then ii) from 18 to 60 polyanioniclayers consisting of hyaluronic acid, or (b2) depositing on the surfaceof said device ii) and then i) as defined above, and optionallyrepeating b1) and/or b2), wherein said polyelectrolyte coating hasantimicrobial activity.
 12. The method for preparing a device accordingto claim 11, further comprising at least one washing after i) and/or ii)of b1) or b2).
 13. The method for preparing a device according to claim11, further comprising at least one drying after i) and/or ii) of b1) orb2) and/or at least one washing after i) and/or ii) of b1) or b2).
 14. Abandage having biocidal activity comprising a polyelectrolyte coatingaccording to claim
 1. 15. The polyelectrolyte coating of claim 1,wherein n is an integer comprised between 25 and
 75. 16. Thepolyelectrolyte coating of claim 1, wherein the n repetitive unitshaving the formula (1) are mobile and/or diffuse within thepolyelectrolyte coating.
 17. The polyelectrolyte coating of claim 1,wherein at least one polycationic layer and at least one polyanioniclayer are not covalently coupled.